Resolving genome fractions using polymorphism counts

ABSTRACT

Methods of reliably estimating genomic fraction (e.g., fetal fraction) from polymorphisms such as small base variations or insertions-deletions are disclosed. Sequenced data from a multigenomic source is used to determine allele counts for one or more of the polymorphisms. For one or more of the polymorphisms, zygosity is assigned, and genomic fraction is determined from the zygosity and allele counts. Certain embodiments employ SNPs as the relevant polymorphism. The disclosed methods can be applied as part of an intentional, pre-designed re-sequencing study targeted against known polymorphisms or can be used in a retrospective analysis of variations found by coincidence in overlapping sequences generated from maternal plasma (or any other setting where a mixture of DNA from several people are present).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ProvisionalApplication Ser. No. 61/474,362, filed Apr. 12, 2011, the contents ofwhich are incorporated herein by reference in their entirety and for allpurposes.

BACKGROUND

The discovery of free floating fetal DNA (sometimes termed “cell freeDNA” or “cfDNA”) in maternal blood allows the possibility of detectingchromosomal abnormality, aneuploidy, and aberration from blood samples.Fractional abundance of fetal DNA in maternal blood plasma is notconstant and varies with a variety of factors including sample handlingand gestational age.

When using DNA sequencing to identify chromosomal aberrations or geneticdefects it is important to know the relative abundance of fetal DNA inthe total population of DNA. For example, when fetal fraction is known,the statistical power (the probability of identifying anomalous cases,or the sensitivity) can be calculated by permutation methods or viaintegration of linear combinations or convolutions of non-central Fdistributions from alpha to infinity where alpha critical point for thesignificance (maximum likelihood of falsely calling an anomaly) of thepopulation of scores under the null hypothesis of no aberration.

A drawback of existing methods for detecting fetal fraction is that theyreply upon measures of the abundance of sex chromosomes (which can onlybe used to reliably measure relative abundance of male embryonic DNA) ormRNA sequence of genes known to be differentially expressed betweenpregnant and embryonic tissue (which is subject to variability ofexpression due to gestational age or other factors).

Estimation of fetal fraction can be difficult because of severalnuisance factors including: parental ethnic differential populationgenetics parameters and sequencing errors. Therefore it is desirable tohave methods robust in the presence of these and other commonlyoccurring confounding factors.

SUMMARY

Certain disclosed embodiments relate to computational methods ofreliably measuring the relative abundance of fetal free floating DNA bysequencing a maternal blood sample.

In specific embodiments, the invention provides methods of reliablyestimating fetal fraction from polymorphisms such as small basevariations or insertions-deletions which are robust with respect toparental ethnicity, embryo sex, gestational age and other environmentalfactors. Many examples disclosed herein employ SNPs as the relevantpolymorphism. The invention can be applied as part of an intentional,pre-designed re-sequencing study targeted against known polymorphismsorcan be used in a retrospective analysis of variations found bycoincidence in overlapping sequences generated from maternal plasma (orany other setting where a mixture of DNA from several people arepresent).

This document presents techniques for the estimation of fractionalabundance of fetal DNA in maternal blood samples. Certain disclosedtechniques use the observed allele frequencies of SNPs found by chanceor found in panels of pre-known SNPs designed for the purpose ofestimating fetal fraction.

While much of the disclosure concerns estimating the fraction of fetalnucleic acid in a sample, the invention is not so limited. Thetechniques and apparatus described herein can be employed in manyinstances to estimate the fraction of nucleic acid from one genome in amixture of two genomes, which may or may not be related as parent andchild genomes.

Certain aspects of the disclosure pertain to methods of estimating thefraction of fetal DNA in DNA obtained from a bodily fluid of a pregnantindividual. Such methods may be characterized by the followingoperations: (a) receiving a sample of the bodily fluid; (b) extractingDNA from the sample under conditions that extract DNA of both a maternalgenome and a fetal genome present in the bodily fluid; (c) sequencingthe extracted DNA with a nucleic acid sequencer under conditions thatproduce DNA segment sequences containing one or more polymorphisms; (d)mapping the DNA segment sequences derived from sequencing the DNA in thebodily fluid to one or more designated polymorphisms on a referencesequence; (e) determining allele frequencies of the mapped DNA segmentsequences for at least one of the designated polymorphisms; (f)classifying the at least one designated polymorphism based on acombination of the zygosity of the pregnant individual and the zygosityof the fetus; and (g) estimating the fraction of fetal DNA in the DNAobtained from the pregnant individual using the allele frequenciesdetermined in (e) and the combination of zygosities from (f).

The mapping may be performed using a computational apparatus programmedto map nucleic acid sequences to the one or more designatedpolymorphisms. In general, any of operations (d)-(g) may be performed onone or more processors running under program instructions.

In certain embodiments, the DNA obtained from a bodily fluid of apregnant individual is cell-free DNA obtained from the plasma of thepregnant individual. Typically, the sequencing is conducted withoutselectively amplifying any of the one or more designated polymorphisms.

In certain embodiments, mapping the DNA segments obtained from the bloodof the individual carrying the fetus comprises computationally mappingthe segments to a database of polymorphisms. In certain embodiments, theclassifying in (f) classifies the at least one designated polymorphisminto one of the following combinations: (i) the pregnant individual ishomozygous and the fetus is homozygous, (ii) the pregnant is individualhomozygous and the fetus is heterozygous, (iii) the pregnant individualis heterozygous and the fetus is homozygous, and (iv) the pregnantindividual is heterozygous and the fetus is heterozygous.

Various filtering operations may be employed. These include, forexample, removing from consideration any polymorphism classified incombination (i) or combination (iv). In another example, the methodsfurther include filtering the at least one designated polymorphisms toremove from consideration any polymorphism having a minor allelefrequency of greater than a defined threshold. In yet another example,the methods include an operation of filtering the at least onedesignated polymorphisms to remove from consideration any polymorphismhaving a minor allele frequency of less than a defined threshold.

The classifying operation may be implemented in various ways. Forexample, it may involve applying a threshold to the allele frequencydetermined in (e). In another example, the classifying operationinvolves applying the allele frequency data from (e), obtained for aplurality of polymorphisms, to a mixture model. In one implementation,the mixture model employs factorial moments.

The fetal fraction determined as described herein may be used forvarious applications. In some examples, the methods described hereininclude an operation of executing program instructions on the one ormore processors to automatically record the fraction of fetal of DNA asdetermined in (g) in a patient medical record, stored on a computerreadable medium, for the pregnant individual. The patient medical recordmay be maintained by a laboratory, physician's office, a hospital, ahealth maintenance organization, an insurance company, or a personalmedical record website. In another application, the estimate of thefraction of fetal DNA is used to prescribe, initiate, and/or altertreatment of a human subject from whom the maternal test sample wastaken. In another application, the estimate of the fraction of fetal DNAis used to order and/or perform one or more additional tests.

Another aspect of the disclosure concerns apparatus for estimating thefraction of fetal DNA in DNA obtained from a bodily fluid of a pregnantindividual. Such apparatus may be characterized by the followingfeatures: (a) a sequencer configured to (i) receive DNA extracted from asample of the bodily fluid comprising DNA of both a maternal genome anda fetal genome, and (ii) sequence the extracted DNA under conditionsthat produce DNA segment sequences containing one or more designatedpolymorphisms; and (b) a computational apparatus configured to (e.g.,programmed to) instruct one or more processors to perform variousoperations such as those described with two or more of the methodoperations described herein. In some embodiments, the computationalapparatus is configured to (i) map nucleic acid sequences to the one ormore designated polymorphisms on a reference sequence, (ii) determineallele frequencies of the mapped DNA segment sequences for at least oneof the designated polymorphisms, (iii) classify the at least onedesignated polymorphism based on a combination of the zygosity of thepregnant individual and the zygosity of the fetus, and (iv) estimate thefraction of fetal DNA in the DNA obtained from the pregnant individualusing the allele frequencies and the combination of zygosities.

In certain embodiments, the apparatus also includes a tool forextracting DNA from the sample under conditions that extract DNA of boththe maternal genome and the fetal genome. In some implementations, theapparatus includes a module configured to extract cell-free DNA obtainedfrom plasma of the pregnant individual for sequencing in the sequencer.

In some examples, the apparatus includes a database of polymorphisms.The computational apparatus may be further configured to instruct theone or more processors to map the DNA segments obtained from the bloodof the individual carrying the fetus by computationally mapping thesegments to the database of polymorphisms. The sequences in the databaseis an example of a reference sequence. Other examples of referencesequences are presented below.

In certain embodiments, the computational apparatus is furtherconfigured to instruct the one or more processors to classify the atleast one designated polymorphism into one of the followingcombinations: (i) the pregnant individual is homozygous and the fetus ishomozygous, (ii) the pregnant is individual homozygous and the fetus isheterozygous, (iii) the pregnant individual is heterozygous and thefetus is homozygous, and (iv) the pregnant individual is heterozygousand the fetus is heterozygous. In some embodiments, the computationalapparatus is further configured to instruct the one or more processorsto remove from consideration any polymorphism classified in combination(i) or combination (iv).

In certain embodiments, the computational apparatus is furtherconfigured to instruct the one or more processors to remove fromconsideration any polymorphism having a minor allele frequency ofgreater than a defined threshold. In some embodiments, the computationalapparatus is further configured to instruct the one or more processorsto filter the one or more designated polymorphisms to remove fromconsideration any polymorphism having a minor allele frequency of lessthan a defined threshold. In certain embodiments, the computationalapparatus is further configured to instruct the one or more processorsto classify the at least one designated polymorphism by applying athreshold to the allele frequency.

In certain embodiments, the computational apparatus is furtherconfigured to instruct the one or more processors to classify the atleast one designated polymorphism by applying the allele frequency dataobtained for a plurality of polymorphisms, to a mixture model. Themixture model may employ factorial moments.

In certain embodiments, the computational apparatus is furtherconfigured to instruct the one or more processors to automaticallyrecord the fraction of fetal of DNA in a patient medical record, storedon a computer readable medium, for the pregnant individual. The patientmedical record may be maintained by a laboratory, physician's office, ahospital, a health maintenance organization, an insurance company, or apersonal medical record website.

Another aspect of the disclosure concerns methods of estimating afraction of fetal DNA in DNA obtained from a bodily fluid of a pregnantindividual according to the following operations: (a) mapping segmentsof DNA obtained from the bodily fluid of the pregnant individual to aplurality of polymorphism sequences, wherein the DNA was sequenced underconditions that identify the plurality of polymorphism sequences; (b)determining an allele frequency of the mapped nucleic acids for each ofthe plurality polymorphism sequences; and (c) applying the allelefrequencies to a mixture model to obtain an estimate of the fraction offetal DNA in the DNA obtained from the blood of the individual carryingthe fetus. Any one or more of operations (a)-(c) may be performed on oneor more processors running under program instructions. In certainembodiments, operation (c) involves executing instructions on the one ormore processors for solving a series of equations for factorial momentsof allele frequency data for each of the plurality of polymorphismsequences. In some embodiments, the mixture model accounts forsequencing error.

In certain embodiments, the methods additionally include computationallyremoving allele frequencies for polymorphisms identified has beingheterozygous in both the fetus and the pregnant individual. In someimplementations, prior to (c), the methods include an operation ofcomputationally removing allele frequencies for polymorphisms identifiedhas being homozygous in both the fetus and the pregnant individual. Insome implementations, prior to (c), the methods include an operation ofcomputationally removing allele frequencies for polymorphisms identifiedhas being heterozygous in the pregnant individual.

The DNA obtained from a bodily fluid of a pregnant individual may becell-free DNA obtained from the plasma of the pregnant individual. Themapping of the nucleic acids obtained from bodily fluid may beimplemented by mapping the segments to a database of polymorphisms.

The methods of this aspect of the disclosure may further includesequencing the DNA from the bodily fluid of pregnant individual with anucleic acid sequencer under conditions that produce DNA segmentsequences containing the polymorphism sequences.

In some implementations, the mapping in (a) comprises identifying aplurality of biallelic polymorphism sequences. In other embodiments, themapping in (a) comprises mapping the segments of DNA to a plurality ofpre-defined polymorphism sequences.

In some embodiments, the methods of this aspect additionally includeexecuting program instructions on the one or more processors toautomatically record the fraction of fetal of DNA as determined in (c)in a patient medical record, stored on a computer readable medium, forthe pregnant individual. The patient medical record may be maintained bya laboratory, physician's office, a hospital, a health maintenanceorganization, an insurance company, or a personal medical recordwebsite.

Based on the estimate of the fraction of fetal DNA, the methods of thisaspect may further include prescribing, initiating, and/or alteringtreatment of a human subject from whom the maternal test sample wastaken. Based on the estimate of the fraction of fetal DNA, the methodsof this aspect may further include ordering and/or performing one ormore additional tests.

In accordance with yet another aspect of the disclosure, methods areprovided for estimating the fraction of fetal DNA in DNA obtained from abodily fluid of a pregnant individual using the following operations:(a) receiving a sample of the bodily fluid; (b) extracting DNA from thesample under conditions that extract DNA of both a maternal genome and afetal genome present in the bodily fluid; (c) sequencing the extractedDNA with a nucleic acid sequencer under conditions that produce DNAsegment sequences; (d) comparing the DNA segment sequences derived fromthe bodily fluid and from the comparison identifying one or morebiallelic polymorphisms; (e) determining allele frequencies of the DNAsegment sequences for at least one of the identified polymorphisms; (f)classifying the at least one identified polymorphism based on acombination of the zygosity of the pregnant individual and the zygosityof the fetus; and (g) estimating the fraction of fetal DNA in the DNAobtained from the pregnant individual using the allele frequenciesdetermined in (e) and the combination of zygosities from (f).

The mapping may be performed using a computational apparatus programmedto map nucleic acid sequences to the one or more designatedpolymorphisms. In general, any of operations (d)-(g) may be performed onone or more processors running under program instructions.

In certain implementations of this aspect, the DNA segment sequenceshave a length of between about 20 base pairs and about 300 base pairs.

In certain embodiments of this aspect, the classifying in (f) classifiesthe at least one identified polymorphism into one of the followingcombinations: (i) the pregnant individual is homozygous and the fetus ishomozygous, (ii) the pregnant is individual homozygous and the fetus isheterozygous, (iii) the pregnant individual is heterozygous and thefetus is homozygous, and (iv) the pregnant individual is heterozygousand the fetus is heterozygous. The methods may further include removingfrom consideration any polymorphism classified in combination (i) orcombination (iv).

In accordance with various embodiments, the methods of this aspect mayinclude filtering and/or classifying operations as described herein inconnection with other aspects. For example, the methods of this aspectmay include filtering the one or more identified polymorphisms to removefrom consideration any polymorphism having a minor allele frequency ofgreater than a defined threshold. In some cases, the classifying the atleast one identified polymorphism includes applying a threshold to theallele frequency determined in (e). The use of mixture models may asdescribed herein may be employed to classify the identifiedpolymorphisms.

Another aspect of the disclosure concerns apparatus for estimating afraction of fetal DNA and including the following elements: (a) asequencer configured to (i) receive DNA extracted from a sample of thebodily fluid comprising DNA of both a maternal genome and a fetalgenome, and (ii) sequence the extracted DNA to produce sequence segmentsof DNA; and (b) a computational apparatus configured to instruct one ormore processors to (i) map the sequence segments of DNA obtained fromthe bodily fluid of the pregnant individual to a plurality ofpolymorphism sequences, (ii) determine an allele frequency for each ofthe plurality of polymorphism sequences from the mapped sequencesegments of DNA, and (iii) apply the allele frequencies to a mixturemodel to obtain an estimate of the fraction of fetal DNA in the DNAobtained from the blood of the individual carrying the fetus.

Yet another apparatus for estimating the fraction of fetal DNA includesthe following elements: (a) a sequencer configured to (i) receive DNAextracted from a sample of the bodily fluid comprising DNA of both amaternal genome and a fetal genome, and (ii) sequence the extracted DNAunder conditions that produce DNA segment sequences; and (b) acomputational apparatus configured to instruct one or more processors to(i) compare the DNA segment sequences derived from the bodily fluid andfrom the comparison identifying one or more biallelic polymorphisms,(ii) determine allele frequencies of the DNA segment sequences for atleast one of the identified polymorphisms, (iii) classify the at leastone identified polymorphism based on a combination of the zygosity ofthe pregnant individual and the zygosity of the fetus, and (iii)estimate the fraction of fetal DNA in the DNA obtained from the pregnantindividual using the allele frequencies and the combination ofzygosities.

The instructions and/or hardware employed in the apparatus aspectsdescribed herein may provide for execution of any one or more of thecomputational or algorithmic operations of the method aspects disclosedherein, regardless of whether such operations are explicitly recitedabove.

These and other features and advantages of the disclosed embodimentswill be described in more detail below with reference to the associateddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram depicting classification of fetal and maternalzygosity states for a given genomic position.

FIG. 2 is an example process flow for implementing some of the disclosedembodiments.

FIG. 3 presents error estimates by sequenced base position over 30 lanesof Illumina GA2 data aligned to human genome HG18 using Eland withdefault parameters.

FIG. 4 is a plot of minor allele count A versus coverage D (assuming noerror) for heterozygosity cases 1 through 4.

FIG. 5 depicts transformation of Case 3 data onto Case 2.

FIG. 6 presents post rotation data, in which D1 was selected so thatcase 1 and cases 2, 3 do not overlap. E1 represents an upper bound ofthe 99^(th) percent upper confidence interval of case 1 data.

FIG. 7 shows a comparison of the results using a mixture model and theknown fetal fraction and estimated fetal fraction.

FIG. 8 shows that using the machine error rate as a known parameterreduces the upward bias by a point.

In FIG. 9 shows that simulated data using the machine error rate as aknown parameter, enhancing the case 1 and 2 error models greatly reducesthe upward bias to less than a point for fetal fraction below 0.2.

FIG. 10 is a schematic depiction of a computer system that, whenappropriately configured (e.g., programmed) or designed, can serve as ananalysis apparatus for disclosed embodiments.

FIGS. 11A and B show a histogram of the number of variant observations(Frequency) at the minor allele percent (A/D) for chromosome chromosomes1(A) and chromosome 7 as produced in an example.

FIGS. 12A and B show the distribution of allelic frequency alongchromosomes 1 (A) and chromosome 7.

DETAILED DESCRIPTION Introduction and Overview

Certain disclosed embodiments involve analyzing DNA taken from apregnant female's blood and using the analysis to estimate the fractionof that DNA that comes from the fetus. The fetal fraction of DNA maythen be used to ascribe some level of confidence to another metric orcharacterization of the fetus based on independent analysis of the DNAtaken from the mother's blood. For example, a fetal DNA sample takenfrom maternal blood may be separately analyzed to detect aneuploidy inthe fetus being carried by the pregnant female. The aneuploidydetermination made by this separate analysis may be given by astatistically grounded confidence level based on the fractional amountof fetal DNA present in the DNA taken from the mother's blood.Relatively low fractions of fetal DNA in the total complement of DNAsuggest a low confidence in any characterization based upon fetal DNA.

Typically, though not necessarily, the analyzed DNA in the mother'sblood is cell-free DNA, although in some embodiments, it may becell-bound DNA. Cell-free DNA is taken from the mother's plasma. Theamount of fetal DNA in the cell-free DNA content taken from pregnantfemales varies widely depending on a variety of factors including thegestational age of the fetus. For typical pregnant human females, it iscurrently believed that about 5-20% of the cell-free DNA is fetal DNA.However, it is not uncommon for the fetal fraction to be significantlylower (e.g., about 1% or lower). In such cases, any separatecharacterization of the fetal DNA may be inherently suspect. On theother hand, some researchers have reported maternal cell-free DNAsamples having fetal DNA fractions as high as 40% or 50%.

In certain implementations described herein, determination of fetalfraction of maternal DNA relies on multiple DNA sequence readings atsequence sites known to harbor one or more polymorphisms. Typically,though not necessarily, such polymorphisms are single nucleotidepolymorphisms (SNP). Other types of suitable polymorphisms includedeletions, STRs (Short Tandem Repeats), insertions, indels (includingmicroindels), etc. Further examples are presented below. In certainembodiments, the polymorphism sites are found on a “reference sequence”as described below. In some embodiments, the polymorphism sites arediscovered while aligning sequence tags to one another and/or areference sequence.

Certain disclosed methods make use of the fact that a fetus's DNAsequences at the polymorphism sites under consideration may notcorrespond to those of its mother. For example, the mother's DNA at thesite of a particular SNP may be homozygous, while the fetus's version ofthe SNP will be heterozygous. Hence, a collection of sequence samplestaken for the SNP in question will be heterogeneous with the majority ofthe sequences containing the major allele and the remaining fractioncontaining the minor allele. The relative amounts of the major and minoralleles are determined by the fraction of fetal DNA in the sample.

It should be mentioned that in a homozygous sample both copies of agiven SNP or other polymorphism contain the same allele, while aheterozygous SNP or other polymorphism contains one copy of the majorallele and one copy of the minor allele. One knows, therefore, that DNAtaken exclusively from a heterozygous individual should contain 50% ofthe major allele and 50% of the minor allele. This knowledge can be usedin elucidating the fraction of fetal DNA as outlined below. As explainedmore fully below, various methods disclosed herein consider onlypolymorphisms in which there are only two alleles in the maternal andfetal DNA, collectively.

In some implementations, the DNA taken from the mother's blood is readmany times, with the total number of reads mapping to a particular siteof a polymorphism being deemed the “coverage” of the polymorphism, andthe number of reads mapping to the minor allele for that polymorphismbeing deemed the minor allele count. The ratio of minor allele count tocoverage is important in various implementations.

Certain of the methods disclosed herein identify and characterize fourcases of polymorphisms in DNA samples that comprise DNA from both themother and the fetus. FIG. 1 below depicts these four cases.Specifically, in a first case, which is rather uninteresting, both themother and the fetus are homozygous at the particular polymorphism beingconsidered. In such case, every sequence in the DNA sample containingthe polymorphism in question will contain the same allele and noinformation can be gleaned about the relative amounts of DNA from themother and the fetus. It should be noted, however, that this case couldbe interesting in the sense that it allows the researcher or technicianto gain some idea of the relative error rate of the DNA sequencingapparatus used to generate the sequence data under consideration.

The second case that the analysis will encounter is a polymorphism forwhich the pregnant female is homozygous and the fetus is heterozygous.In this case, a relatively small, but nevertheless significant, fractionof the detected sequences will contain the minor allele. Specifically,in this second case, the frequency of the minor allele is nominallygiven by the fraction of fetal DNA in the mother's bloodstream dividedby two.

In a third case, the polymorphism under consideration is heterozygous inthe mother's DNA and homozygous in the fetus's DNA. In this situation,the frequency of the minor allele is nominally given by 0.5 minusone-half of the fraction of fetal DNA in the DNA sample.

Finally, in the fourth case, the polymorphism under consideration isheterozygous in both the mother and the fetus. In this case, it isexpected that the frequency of the major and minor alleles will both be0.5. As with the first case, the fourth case is relatively uninterestingfor determining the fetal fraction of DNA.

If the researcher, technician, or software tasked with determining thefraction of fetal DNA in a sample knew for a given polymorphism which ofthe four cases that polymorphism belonged to, then the fraction of fetalDNA could be directly estimated, assuming that the polymorphism underconsideration fell into either case two or case three. In practice,however, one never has this knowledge a priori. Therefore computationalapparatus is required to perform the operations described herein.

In certain embodiments, described elsewhere herein, a thresholdingtechnique is employed to classify a single polymorphism into one of thefour cases. Once the polymorphism is so classified, and found to residein either case 2 or 3, the fetal fraction can be estimated. In otherembodiments, the technique considers multiple polymorphisms distributedthroughout all or a portion of the genome. As illustrated in thespecific examples, multiple different SNPs across the genome may be usedfor this purpose.

In particular embodiments, the allele frequency is determined for anumber of different polymorphisms in a DNA sample taken from a mother'sblood sample. For this plurality of polymorphisms, some fraction willcorrespond to zygosity case 1, another fraction will correspond to case2, a third fraction will correspond to case 3, and a final fraction willcorrespond to case 4. These fractions will sum to a value of 1. Amixture model or related technique may be employed to tease out one ormore statistical properties of the polymorphisms in each of these fourcategories. Specifically, a mixture model may be employed to determine amean and optionally the variance for each of the four cases encounteredin a DNA sample taken from a pregnant female's blood. In specificembodiments, this is the mean and variance associated with the frequencyof the minor allele in relation to the total number of counts for apolymorphism under consideration (coverage). As elaborated elsewhereherein, the mean values for each of these four categories, or at leastthe second and third categories, are directly related to the fetalfraction in the DNA taken from the mother's blood.

In a specific implementation employing mixture models, one or morefactorial moments are calculated for each position where a polymorphismis being considered. For example, a factorial moment (or a collection offactorial moments) is calculated using multiple SNP positions consideredin the DNA sequence. As shown in equation 4 below, each of the variousfactorial moments is a summation over all the various SNP positionsunder consideration for the ratio of minor allele frequency to coveragefor a given position. As shown in equation 5 below, these factorialmoments are also related to the parameters associated with each of thefour zygosity cases described above. Specifically, they related to theprobability for each of the cases as well as the relative amounts ofeach of the four cases in the collection of polymorphisms underconsideration. As explained, the probability is a function of thefraction of fetal DNA in the cell-free DNA in the mother's blood. Asexplained more fully below, by calculating a sufficient number of thesefactorial moments (which are shown in equation 4), the method provides asufficient number of expressions to solve for all the unknowns. Theunknowns in this case would be the relative amounts of each of the fourcases in the population of polymorphisms under consideration as well asthe probabilities (and hence fetal DNA fractions) associated with eachof these four cases. See equation 5. Similar results can be obtainedusing other versions of mixture models as represented in equations 7-12below. These particular versions make use of only polymorphisms fallinginto cases 1 and 2, with polymorphisms for cases 3 and 4 being filteredby a thresholding technique.

Thus, the factorial moments may be used as part of a mixture model toidentify the probabilities of any combination of the four cases ofzygosity. And, as mentioned, these probabilities, or at least those forthe second and third cases, are directly related to the fraction offetal DNA in the total cell-free DNA in the mother's blood.

It should also be mentioned that sequencing error may be employed toreduce the complexity of the system of factorial moment equations thatmust be solved. In this regard, it should be recognized that thesequencing error actually can have any one of four results(corresponding to each of the four possible bases at any givenpolymorphism position).

In certain embodiments, tags are aligned with a reference chromosome orgenome, and biallelic polymorphisms are identified. These polymorphismsare not predefined or otherwise identified prior to alignment. They aresimply identified during alignment and then characterized based on theirzygosities and minor allele counts as described herein. This informationis used to estimate genome fractions as described herein.

The lengths of the tags used in embodiments described herein willgenerally be determined by the sequencing method employed to generatethe tags. The methods are robust across a wide range of tag lengths. Incertain implementations, the tags are between about 20 to 300 base pairsin length (or about 30 to 100 base pairs in length).

An example process flow for implementing some of the disclosedembodiments is shown in FIG. 2. As depicted there, the process begins at201 with collection of DNA (cell-free or cell bound) from maternal bloodor other bodily fluid. From this DNA multiple sequences mapped to one ormore polymorphisms in a reference sequence. This mapping provides anallele frequency for each of the polymorphisms. See block 203.

More specifically, the process at block 203 may involve readingsequences of the collected DNA at locations of multiple polymorphisms.In some cases, these may be generated as part of the process for ploidydeterminations or other determination made with respect to the fetalDNA. Thus, in some embodiments, separate sequences need not begenerated. The read sequences are aligned to a reference sequence tomaximize alignment using BLAST or a similar tool.

The reference sequence may be provided as a database of polymorphisms.In some cases, this is an allele search reference set produced from acombinatorial expansion of all polymorphism definitions (e.g., in thecase where the polymorphisms are SNPs, all SNP sequences). See theappendix for example. In a specific example, the sequences are about 100to 150 base pairs in length.

Returning to FIG. 2, the method determines the maternal/fetal zygositycombination for one or more the polymorphisms considered in theoperation of block 203. See block 205. A mixture model may be employedfor this purpose in certain embodiments. As mentioned, the combinationsare as follows: M&F homozygous, M homozygous and F heterozygous, Mheterozygous and F homozygous, and M&F heterozygous.

Finally, as illustrated at block 207, the method uses the combination ofzygosity case allele frequency at one or more of the polymorphisms toestimate the fractional amount of fetal component in the DNA from thematernal sample.

DEFINITIONS

The following discussion is provided as an aid in understanding certainaspects and advantages of the disclosed embodiments.

The term “read” refers to a sequence read from a portion of a nucleicacid sample. Typically, though not necessarily, a read represents ashort sequence of contiguous base pairs in the sample. The read may berepresented symbolically by the base pair sequence (in ATCG) of thesample portion. It may be stored in a memory device and processed asappropriate to determine whether it matches a reference sequence ormeets other criteria. A read may be obtained directly from a sequencingapparatus or indirectly from stored sequence information concerning thesample.

The term “tag” also refers to short sequences from a nucleic acidsample. Typically, a tag contains associated information such as thelocation of the sequence in the genome. For some purposes, the termsread and tag are interchangeable herein. Typically, however, sequencereads are aligned to a reference sequence, and reads that map at onlyone site on the reference genome are called tags. The “segment sequence”is sometimes used herein interchangeably with “tag.”

Frequently herein “reads” are described as sequences of nucleic acidsthat are 36 base pairs in length (36mers). Of course, the disclosedembodiments are not limited to this size. Smaller and larger reads aresuitable in many applications. For applications that align reads to thehuman genome, a read of size 30 base pairs or greater is generallyconsidered sufficient to map a sample to a single chromosome. Muchlarger tags/reads are suitable for some applications. With whole genomesequencing, reads on the order of 1000 base pairs or greater may beused. In certain embodiments, a read may have a length of between about20 and 10,000 base pairs, or between about 30 and 1000 base pairs, orbetween about 30 and 50 base pairs.

A “reference sequence” is a sequence of a biological molecule, which isfrequently a nucleic acid such as a chromosome or genome. Typicallymultiple reads are members of a given reference sequence. In certainembodiments, a read or tag is compared to a reference sequence todetermine whether the reference sequence contains the read sequence.This process is sometimes referred to as alignment.

In various embodiments, the reference sequence is significantly largerthan the reads that are aligned to it. For example, it may be at leastabout 100 times larger, or at least about 1000 times larger, or at leastabout 10,000 times larger, or at least about 10⁵ times larger, or atleast about 10⁶ times larger, or at least about 10⁷ times larger.

In one example, the reference sequence is that of a full length humangenome. Such sequences may be referred to as genomic referencesequences. In another example, the reference sequence is limited to aspecific human chromosome such as chromosome 13. Such sequences may bereferred to as chromosome reference sequences. Other examples ofreference sequences include genomes of other species, as well aschromosomes, sub-chromosomal regions (such as strands), etc. of anyspecies.

In various embodiments, the reference sequence is a consensus sequenceor other combination derived from multiple individuals. However, incertain applications, the reference sequence may be taken from aparticular individual.

The term “alignment” refers to the process of comparing a read or tag toa reference sequence and thereby determining whether the referencesequence contains the read sequence. If the reference sequence containsthe read, the read may be mapped to the reference sequence or, incertain embodiments, to a particular location in the reference sequence.In some cases, alignment simply tells whether or not a read is a memberof a particular reference sequence (i.e., whether the read is present orabsent in the reference sequence). For example, the alignment of a readto the reference sequence for human chromosome 13 will tell whether theread is present in the reference sequence for chromosome 13. A tool thatprovides this information may be called a set membership tester. In somecases, an alignment additionally indicates a location in the referencesequence where the read or tag maps to. For example, if the referencesequence is the whole human genome sequence, an alignment may indicatethat a read is present on chromosome 13, and may further indicate thatthe read is on a particular strand of chromosome 13.

A “site” is a unique position in a reference sequence corresponding to aread or tag. In certain embodiments, it specifies the identity of achromosome (e.g., chromosome 13), a strand of the chromosome, and anexact position in the chromosome.

“polymorphic site” is a locus at which nucleotide sequence divergenceoccurs. The locus may be as small as one base pair. Illustrative markershave at least two alleles, each occurring at frequency of greater than1%, and more typically greater than 10% or 20% of a selected population.A polymorphic site may be as small as one base pair. The terms“polymorphic locus” and “polymorphic site” are herein usedinterchangeably.

“polymorphic sequence” herein refers to a nucleic acid sequence e.g. aDNA sequence, that comprises one or more polymorphic sites e.g one SNPor a tandem SNP. Polymorphic sequences according to the presenttechnology can be used to specifically differentiate between maternaland non-maternal alleles in the maternal sample comprising a mixture offetal and maternal nucleic acids.

DETAILED EMBODIMENTS

Typically, the processes described herein employ a reference sequencethat spans one or more polymorphisms and is associated with the DNAbeing sampled. A reference sequence may be, for example, the humangenome, a chromosome, or a region in a chromosome. One or more of thepolymorphisms can be designated for the purpose of estimating fetal DNAfraction. Polymorphisms that are designated for use in determining fetalfraction are polymorphisms that are pre-known. For example, acomprehensive listing of references, facts and sequence information onpre-known STRs, and related population data are compiled in STRBase,which may be accessed via the World Wide Web atibm4.carb.nist.gov:8800/dna/home.htm. Sequence information from GenBank®(http://www2.ncbi.nlm.nih.gov/cgi-bin/genbank) for commonly used STRloci is also accessible through STRBase. Information of pre-known SNPscan be accessed via are available from publicly accessible databasesincluding, but not limited to Human SNP Database at world wide webaddress wi.mit.edu, NCBI dbSNP Home Page at world wide web addressncbi.nlm.nih.gov, world wide web address lifesciences.perkinelmer.com,Applied Biosystems by Life Technologies™ (Carlsbad, Calif.) at worldwide web address appliedbiosystems.com, Celera Human SNP database atworld wide web address celera.com, the SNP Database of the GenomeAnalysis Group (GAN) at world wide web address gan.iarc.fr. In oneembodiment, the SNPs designated for determining fetal fraction areselected from the group of 92 individual identification SNPs (IISNPs)described by Pakstis el al. (Pakstis et el. Hum Genet127:315-324[2010]), which have been shown to have a very small variationin frequency across populations (F_(st)<0.06), and to be highlyinformative around the world having an average heterozygosity≧0.4. SNPsthat are encompassed by the method of the invention include linked andunlinked SNPs. To designate suitable tandem SNP sequences, theInternational HapMap Consortium database can be searched (TheInternational HapMap Project, Nature 426:789-796[2003]). The database isavailable on the world wide web at hapmap.org.

The polymorphisms so employed may be panels of pre-known polymorphismsdesignated for determining fetal DNA fraction or they may be found bychance in an analysis of maternal DNA for other purposes such as mappingsample DNA tags to chromosomes.

In certain embodiments, the method comprises sequencing DNA in a sampleusing a mixture of genomes e.g. a maternal sample comprising fetal andmaternal cell-free DNA, to provide a plurality of sequence tags that mapto sequences comprising pre-known polymorphic sites on a referencegenome, and using the tags mapped at the pre-known sites to determinethe fetal fraction as described in detail below. Alternatively,following sequencing of the DNA, the sequence tags that are obtained bythe sequencing technology e.g. NGS, are mapped to a reference genome,e.g. hg19, and sequence tags mapping to sites at which polymorphismsoccur by chance i.e. not pre-known, are used to determine the fetalfraction.

The reference sequence to which sequence tags are mapped to pre-knownpolymorphic sites, can be a published reference genome or it can be anartificial database or other pre-defined collection of sequences for thepolymorphisms under consideration. Each of the database sequences willspan the one or more nucleotides associated with the polymorphism. Asone example, see the list of polymorphism sequences presented below in“Appendix 1.”

In various embodiments, the number of polymorphisms employed to estimatefetal DNA fraction is at least 2 polymorphisms, and more particularlyfor each of at least about 10 polymorphisms, and more preferably foreach of at least about 100 polymorphisms.

In one example, SNP coverage and allele frequency are determined byaligning generated sequences to a reference genome constructed fromcombinatory expansion of the SNP definitions. The amplicon databasecontains bi-allelic variation information surrounded by, e.g., at leastabout 50 bases of flanking sequence. For example an amplicon withvariation information string “[g/c]” (representing alternate alleles “g”and “c” may look like:

atcg . . . accg[g/c]ccgt . . .

In some cases, the procedure to input the amplicon database andgenerated sequences and output SNP/allele counts is as follows.

-   -   1. Create an allele search reference set from combinatorial        expansion of the SNP definitions. For each sequence in the        amplicon database, for each allele in the variation information        string, create an allele sequence with the variation information        string replaced by the allele.        -   a. For example, considering the above example amplicon            sequence two sequences would be created: 1) atcg . . .            accgGccgt . . . And 2) atcg . . . accgCccgt . . . .        -   b. An example of a full allele search reference set can be            found in the Allele Search Database Sequence Listing.    -   2. Map sequences to the allele search reference set keeping only        mappings that match only one sequence in the search set.    -   3. Allele count is determined by counting the number of        sequences that match its allele sequence.

The methods disclosed herein assume a “normal” pregnancy, i.e., apregnancy in which the mother is carrying only one fetus, and not twins,triplets, etc. Those of skill will appreciate modifications whichaccount for non-normal pregnancies, particularly those in which thenumber of fetuses is known.

As indicated, when determining fetal fraction, the method sequences theDNA in the sample from maternal blood and counts the sequence tags thatmap to each sequence of polymorphism(s) under consideration. For eachpolymorphism, the method tallies the total number of reads that map toit (the coverage) and the numbers of sequence tags associated with eachallele (the allele counts). In a simple example, a polymorphism having acoverage of 5, may have 3 reads of allele B and 2 reads of allele A. Inthis example, allele A is deemed the minor allele and allele B is deemedthe major allele.

In some embodiments, this operation makes use of very fast sequencingtools such as massively parallel DNA sequencing tools. Examples of suchtools are described in more detail below. In some cases, many thousandsor millions of tag sequences are read for a single sample. Preferably,sequencing is performed in a manner that allows quick and directassignment of sequenced DNA to particular predefined sequences harboringpolymorphisms under consideration. Generally, there is sufficientinformation for this purpose in tags of size 30 base pairs or greater.Tags of this size can be unambiguously mapped to sequences of interest.In a specific embodiment, the tag sequences employed in the process are36 base pairs in length.

The tags are mapped to a reference genome or to sequences in an allelesequence database (e.g., see Appendix 1 as previously mentioned) and thenumber of tags so mapped is determined. This will provide both thecoverage and minor allele count for each polymorphism underconsideration. In some cases, this may be done concurrently with mappingeach tag to one of the 23 human chromosomes and determining the numberof mapped tags per chromosome.

As mentioned, coverage is the total number of read sequences that map toa given polymorphism in a reference sequence. Allele count in the totalnumber of read sequences mapping to such polymorphism that have anallele. The sum of all allele counts must equal the coverage. The allelewith the highest count is the major allele, and the allele with thelowest count is the minor allele. In certain embodiments, the onlyinformation needed to estimate fetal DNA fraction is the coverage andminor allele count for each of a plurality of polymorphisms. In someembodiments, a base calling error rate of the DNA sequencing apparatusis also used.

It is useful to consider the mathematical or symbolic underpinnings ofcertain methods disclosed herein. As mentioned, in various examples,sequences generated from maternal blood are aligned (superimposed sothat identical bases are maximized) to a reference genome or othernucleic acid sequence. Given a genomic position, j, and a set ofsequences aligned to the reference, let the number of occurrences ofeach of the four DNA bases (“a”, “t”, “g”, and “c”, also called“alleles”), among the aligned sequences be w(j,1), w(j,2), w(j,3), andw(j,4) respectively. For the purposes of this discussion one may assumewithout loss of generality that all variations are bi-allelic. Hence thefollowing notations may be used:

Major Allele Count

at genomic position j as

-   -   B≡B_(i)≡{b_(j)}≡w_(j,i) ⁽¹⁾=max_(iε{1,2,3,4}){w_(j,i)} as the        first order statistic of counts at position j (The major allele,        b, is the corresponding argmax. Subscripts are used when more        than one SNP is being considered.),

Minor Allele Count

-   -   at position j as A≡A_(i)≡{a_(j)}=w_(j,i) ⁽²⁾ as the second order        statistic of counts (i.e. the second highest allele count) at        position j,

Coverage

at position j as D≡D_(j)={d_(i)}=A_(j)+B_(j), and

Sequencing machine Error Rate

is denoted e.

When the context is clear, for convenience notations are usedinterchangeably; for example, A, Ai, or {ai} may be used interchangeablyfor the minor allele or the minor allele count. Subscripts may or maynot be used depending if more than one SNP is being considered. (SNPsare used for purposes of example only. Other types of polymorphisms maybe used as discussed elsewhere herein.).

In FIG. 1, the basis for the four states of polymorphism zygosity isdepicted. As illustrated, the mother may be homo or heterozygous at agiven polymorphism. Similarly, the baby may be either heterozygous orhomozygous at the same position. As illustrated, cases 1 and 2 are thepolymorphism cases in which the mother is homozygous. If the baby andthe mother are both homozygous, the polymorphism is a case 1polymorphism. As indicated above, this situation is typically notparticularly interesting. If the mother is homozygous and the baby isheterozygous, the fetal fraction, f, is nominally given by two times theratio of the minor allele to the coverage. In the polymorphism casewhere the mother is heterozygous and the baby is homozygous (case 3 inFIG. 1), the fetal fraction is nominally one minus two times the ratioof the minor allele to the coverage. Finally, in the case where both themother and the fetus are heterozygous, the minor allele fraction shouldalways be 0.5, barring error. The fetal fraction cannot be derived forpolymorphisms falling into case 4.

The four cases will now be further elaborated.

Case 1: Mother and Baby Homozygous

-   -   In this case, baring sequencing error or contamination, no        differences should be observed.

E(min allele frequency)=E(A)=0.

-   -   In practice A˜(is distributed as) a Binomial distribution which        is approximated well by the Poisson distribution for low np. The        distribution rate parameter for Binomial or Poisson is related        to the sequencing error rate, e and the coverage D. FIG. 3 shows        mismatch frequencies of generated 36mer sequences aligned to a        Human reference genome.    -   This case contains no information about the fetal fraction.

FIG. 3 presents error estimates by sequenced base position over 30 lanesof Illumina GA2 data aligned to Human Genome HG18 using Eland withdefault parameters.

Case 2: Mother Homozygous and Baby Heterozygous

-   -   In this case, for small fetal fraction (f), the observed allele        frequencies will be markedly different. With the major allele        usually occurring at frequency several times more than the minor        allele.    -   Barring error, given a single SNP position (D,A), E(A)=Df/2 and        an unbiased estimate for f is 2A/D    -   Barring error, A˜Binomial(f/2,D). Mean Df/2, Variance        (1−f/2)Df/2. [Approximately Normal dist if D>15].

Case 3: Mother Heterozygous and Baby Homozygous

-   -   In this case the observed frequencies for major and minor        alleles are close and A/D is just under 0.5.    -   Barring error, E(A)=D(1−f)/2, and E(1−(2A/D))=f    -   Barring error, A˜Binomial((1−f)/2, D). Mean D((1−f)/2), Variance        D/4(1−f̂2).

Case 4: Mother Heterozygous and Baby Heterozygous

Note that, barring error, there are two sub-cases for this.

Case 4.1: The Allele from the Father is Different from the Mother'sAlleles

-   -   This would introduce a third allele which would be the minor        allele with E(A)=Df/2. These cases should not have an effect        upon estimations for f because the procedure for assigning        sequences to amplicons will filter out these cases when the        reference SNPs are bi-allelic.

Case 4.2: The Allele from the Father Matches One of the Mother's Alleles

-   -   In this case, barring error, the two alleles would appear at 1:1        proportion so that this case is not useful for fetal fraction        estimation.    -   Barring error, E(A)=0.5, and A˜Binomial(0.5,D) truncated at 0.5.

FIG. 4 presents a plot of minor allele count A versus coverage D(assuming no error) for heterozygosity cases 1 through 4.

In various embodiments, the method broadly concerns analyzing the allelefrequency at one or more SNPs (or other polymorphisms) to classify thepolymorphisms as being in either case 2 and/or case 3. Using the allelefrequency in conjunction with the classification, the method canestimate the fetal fraction.

In some cases, given minor allele count A and coverage D, in other wordsa single point (D,A), for an individual SNP position allows methods tomake a single point estimate. For example, certain methods classify aSNP with allele count (D,A) into a single case and derive a fetalfraction estimate as follows:

ES1.1 Simple Thresholds to Decide Case

Given an individual position (SNP),

-   -   1. Decide on case 1 with a decision function like 2A/D<e or a        defined critical value of Binomial(e,D) or Poisson(De). One may        also use an alternative distribution within the scope of this        invention). No fetal fraction (f) estimate.    -   2. Decide on case 4 if 2A/D>(0.5−e) or some critical value of        Binomial(0.5,D), (or other suitable approximating distribution).        Do not use the position for an estimate of f.    -   3. Otherwise, decide on case 2 if 2A/D<0.25 (or some other        manually set or automatically estimated threshold). Fetal        fraction f estimated as 2A/D    -   4. Otherwise case 3. Use fetal fraction estimate f=(1−2A/D).

Accuracy can be gained by combining allele count information fromseveral SNPs to estimate fetal fraction.

Method EM1: Combine Multiple SNPs by Averaging.

-   -   Take mean, median, other center measurement (for example: Tukey        bi-weight, M-estimators, etc. . . . ). Weighted averages may        also be used. For an example of how weights may be defined see        EM2.4 below. Additionally robust measures of center may be used.        Method EM2 Simultaneous Estimation from Case 2 and Case 3 by        Transformation    -   For occasions where f is less than X % case 3 points (D,A) can        be transformed to be coincident with case 2 points. From this        line a common slope can be calculated via regression through the        origin (see FIG. 5).    -   One theoretical drawback of methods based on transformation is        the case 2 and 3 binomial distributions will have different        shape. At typical fetal fraction levels (<10%) case 2 data will        have a distribution close to Poisson skewed to the right and        case 3 will have a distribution close to normal.

FIG. 5 depicts the transformation of Case 3 data onto Case 2. Now asingle regression can estimate f from both cases simultaneously.

Method for Calculating EM2.3:

Step 1: Throw out Case 4 Data

-   -   For each data point (D,A) if A>(0.5D−T1) then exclude (D,A) from        further analysis. T1(D,A) a real valued function.

Step 2: Transform Case 3 Data

-   -   See FIG. 6. For each data point (D,A) not ruled to be 4, if        A>T2*D then transform to points to new coordinates (D1,A1).        T2(D,A) a real valued function.

α=2A/D

A1=−1(0.5i−A)

D1=D

Step 3: Establish Threshold DT to Reduce Pollution from Case 1 Data

-   -   Disregard all data points below T2(D,A) a real valued function.

Step 4: Regression Estimate for Remaining Transformed Case 2 and 3 Data.

-   -   Apply regression through origin to remaining points. Fetal        fraction estimate is twice the slope of the regression like.

Note that there are many classes of transformations that can beconstructed to accomplish the same coincidence of case 2 and 3 data.Examples include trigonometric, transformation or use of rotationmatrices. These deviations are intended to be included in the scope ofthis disclosure. Furthermore, many classes of regression (L2, L1, . . .) or optimization can be used. Swapping the optimization algorithm is atrivial change and covered under the scope of this disclosure.

FIG. 6 presents post rotation data. Selecting D1 so that case 1 andcases 2 and 3 do not overlap. E1 represents an upper bound of the99^(th) percent upper confidence interval of case 1 data.

Method EM3 Weighted Least Squares

The regression method from EM2.3 assumes all of the translated datapoints have equal variance. It is more proper to account for theheteroskedasticity of the different data sources and even of points fromthe same heterozygosity pattern.

Steps 1 through 3 are identical to EM2.3.

Step 4: Regression

-   -   In the regression from EM2.3, the points from case 2 data will        have variance v2(f,D)=[0.5*Df−0.25*Df̂2] and points from case 3        data will have variance v3(f,D)=[0.25D(1−f̂2)]. Assuming we give        each point a different weight, w, as in EM2.3, we seek to        minimize

$\begin{matrix}{Q = {\sum\limits_{i = 1}^{n}\; {w_{i}\left( {a_{i} - {sd}_{i}} \right)}^{2}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

Setting first derivatives to zero and solving for s:

$\begin{matrix}{{{\frac{\partial Q}{\partial s} = {{\sum\limits_{i = 1}^{n}\; {2{w_{i}\left( {d_{i} - {sa}_{i}} \right)}\left( {- a_{i}} \right)}} = 0}}{{{\sum\limits_{i = 1}^{n}\; {sa}_{i}^{2}} - {\sum\limits_{i = 1}^{n}\; {2w_{i}a_{i}x_{i}}}} = 0}{and}{s = \frac{\sum\limits_{i = 1}^{n}\; {2w_{i}d_{i}a_{i}}}{\sum\limits_{i = 1}^{n}\; a_{i}^{2}}}{{where}\mspace{14mu} d_{i}\mspace{14mu} {is}\mspace{14mu} {the}\mspace{14mu} {coverage}\mspace{14mu} {of}\mspace{14mu} {SNP}\mspace{14mu} i\mspace{14mu} {and}\mspace{14mu} a_{i}\mspace{14mu} {is}\mspace{14mu} {{the}\left( {{transformed}\mspace{14mu} {for}\mspace{14mu} {case}\mspace{14mu} 3} \right)}}{{minor}\mspace{14mu} {allele}\mspace{14mu} {count}\mspace{14mu} {of}\mspace{14mu} {SNP}\mspace{14mu} {i.}}}} & {{Equation}\mspace{14mu} 2}\end{matrix}$

This method weights with the inverse of the variance of each point,estimated as v2(2A/D,D), or v3(2A/D,D) as appropriate. The fetalfraction estimate is 2*s.

In certain embodiments, a mixture model may be employed to classify acollection of polymorphisms into two or more of the zygosity cases andconcurrently estimate the fetal DNA fraction from mean allelefrequencies for each of these cases. Generally, a mixture model assumesthat a particular collection of data is made up of a mixture ofdifferent types of data, each of which has its own expected distribution(e.g., a normal distribution). The process attempts to find the mean andpossibly other characteristics for each type of data. In embodimentsdisclosed herein, there are up to four different data types (thezygosity cases) that make the up the minor allele frequency data for thepolymorphisms under consideration.

One implementation of a mixture model is presented in the followingsection. In this embodiment, the minor allele frequency A is a sum offour terms as shown in equation 3. Each of the terms corresponds to oneof the four zygosity cases. Each term is the product of a polymorphismfraction and a binomial distribution of the minor allele frequency. Theas are the fractions of the polymorphisms falling into each of the fourcases. Each binominal distribution has an associated probability, p, andcoverage, d. The minor allele probability for case 2, for example, isgiven by f/2.

The disclosed embodiments make use of “factorial moments” for the allelefrequency data under consideration. As is well known, a distribution'smean is the first moment. It is the expected value of the minor allelefrequency. The variance is the second moment. It is calculated from theexpectation value of the allele frequency squared.

The allele frequency data across all polymorphisms may be used tocalculate factorial moments (a first factorial moment, a secondfactorial moment, etc.) as shown in equation 4. As indicated by theseequations, the factorial moments are summations of terms over i, theindividual polymorphisms in the data set, where there are n suchpolymorphisms in the data set. The terms being summed are functions ofthe minor allele counts, a_(i), and coverages d_(i).

Usefully, the factorial moments have relationships with the values ofα_(i) and p_(i) as illustrated in equation 5. From the probabilities,p_(i), one can determine the fetal fraction, f. For example, p₂=f/2 andp₃ is 1−f/2. Thus, the responsible logic can solve a system of equationsrelating the unknown αs and ps to the factorial moment expressions forminor allele fractions across the multiple polymorphisms underconsideration. Of course, there are other techniques for solving themixture models within the scope of this invention.

It is useful to further consider the mathematical or symbolicunderpinnings of mixture model embodiments disclosed herein. The fourheterozygosity cases described above suggest the following Binomialmixture model for the distribution of ai in points (ai,di):

A={a _(i)}˜α₁Bin(p ₁ ,d _(i))+α₂Bin(p ₂ ,d _(i))+α₃Bin(p ₃ ,d_(i))+α₄Bin(p ₄ ,d _(i))

where

1=α₁+α₂+α₃+α₄

m=4

Equation 3

Various models for relating the p_(i) to fetal fraction and sequencingerror rates are described below. The parameters α_(i) relate topopulation specific parameters and the ability to let these values“float” give these methods additional robustness with respect to factorslike ethnicity and progeny of the parents.

For various heterozygosity cases the equation above can be solved forfetal fraction. Perhaps the easiest method to solve for fetal fractionis through the method of factorial moments in which the mixtureparameters can be expressed in terms of moments that can easily beestimated from the observed data.

Given n SNP positions, Factorial moments are defined as follows:

$\begin{matrix}{{F_{1} = {\frac{1}{n}{\sum\limits_{i = 1}^{n}\; \frac{a_{i}}{d_{i}}}}}{F_{2} = {\frac{1}{n}{\sum\limits_{i = 1}^{n}\; \frac{a_{i}\left( {a_{i} - 1} \right)}{d_{i}\left( {d_{i} - 1} \right)}}}}\ldots {F_{j} = {\frac{1}{n}{\sum\limits_{i = 1}^{n}\; \frac{{a_{i}\left( {a_{i} - 1} \right)}{\cdots \left( {a_{i} - j + 1} \right)}}{{d_{i}\left( {d_{i} - 1} \right)}\left( {d_{i} - j + 1} \right)}}}}} & {{Equation}\mspace{14mu} 4}\end{matrix}$

Factorial moments can be related to the {α_(i),p_(i)} with

$\begin{matrix}{{F_{1} \approx {\sum\limits_{i = 1}^{m}\; {\alpha_{i}p_{i}^{1}}}}{F_{2} \approx {\sum\limits_{i = 1}^{m}\; {\alpha_{i}p_{i}^{2}}}}\ldots {F_{j} \approx {\sum\limits_{i = 1}^{m}\; {\alpha_{i}p_{i}^{j}}}}\ldots {F_{g} \approx {\sum\limits_{i = 1}^{m}\; {\alpha_{i}p_{i}^{g}}}}} & {{Equation}\mspace{14mu} 5}\end{matrix}$

A solution can be identified by solving for the {α_(i),p_(i)} in asystem of equations derived from the above relation Equation 5 whenn>2*(number of parameters to be estimated). Obviously, the problembecomes much more difficult mathematically for higher g as more{α_(i),p_(i)} need to be estimated.

It is typically not possible to accurately discriminate between case 1and 2 (or case 3 and 4) data by simple thresholds at lower fetalfractions. Luckily for the use of reduced case models, case 1/2 data iseasily separated from case 3/4 data by discriminating at point (2A/D)=T.Use of T=0.5 has been found to perform satisfactorily.

Note that the mixture model method employing equations 4 and 5 makes useof the data for all polymorphisms but does not separately account forthe sequencing error. Appropriate methods that separate data for thefirst and second cases from data for the third and fourth cases canaccount for sequencing error.

In further examples, the data set provided to a mixture model containsdata for only case 1 and case 2 polymorphisms. These are polymorphismsfor which the mother is homozygous. A threshold technique may beemployed to remove the case 3 and 4 polymorphisms. For example,polymorphisms with minor allele frequencies greater than a particularthreshold are eliminated before employing the mixture model. Usingappropriately filtered data and factorial moments as reduced toequations 7 and 8, one may calculate the fetal fraction, f, as shown inequation 9. Note that equation 7 is a restatement of equation 3 for thisimplementation of a mixture model. Note also that in this particularexample, the sequencing error associated with the machine reading is notknown. As a consequence, the system of equations must separately solvefor the error, e.

FIG. 7 shows a comparison of the results using this mixture model andthe known fetal fraction (x axis) and estimated fetal fraction. If themixture model perfectly predicted the fetal fraction, the plottedresults would follow the dashed line. Nevertheless, the estimatedfractions are remarkably good, particularly considering that much of thedata was eliminated prior to applying the mixture model.

To further elaborate, several other methods are available for parameterestimation of the model from Equation 3. In some cases tractablesolution can be found by setting derivates to zero of the chi-squaredstatistic. In cases where no easy solution can be found by directdifferentiation, taylor series expansion of the binomial PDF or otherapproximating polynomials can be effective. Minimum chi-squareestimators are well-known to be efficient.

$\begin{matrix}{{\chi^{2}\left( {\alpha_{i},p_{i}} \right)} = {\sum\limits_{i = 1}^{n}\; \frac{\left( {P_{i} - {\sum{\alpha_{i}{{Binomial}\left( {p_{i},{di}} \right)}}}} \right)^{2}}{{Binomial}\left( {n,p} \right)}}} & {{Equation}\mspace{14mu} 6}\end{matrix}$

Where Pi is the number of points of count i. An alternative method fromLe Cam [“On the Asymptotic Theory of Estimation and Testing Hypotheses”Proceedings of the Third Berkeley Symposium on Mathematical Statisticsand Probability, Volume 1 Berkeley Calif.: University of CA Press, 1956,pp. 129-156] uses Ralph-Newton iteration of the likelihood function. Themethod of moments solutions from Equation 5 can be used as a startingpoint for the iteration.

Under another application a method of resolving mixture models involvingexpectation maximization methods operating on mixtures of approximatingBeta distributions is discussed.

Model Cases (1+2), Sequencing Error Unknown

Consider a reduced model that only accounts for heterozygosity cases 1and 2. In this case the mixture distribution can be written as

A={a _(i)}˜α_(i)Bin(e,d _(i))+α₂Bin(f/,2,d _(i))

where

1=α₁+α₂

m=4

Equation 7

And the system

F ₁=α₁ e+(1−α₁)(f/2)

F ₂=α₁ e ²+(1−α₁)(f/2)²

F ₃=α₁ e ³+(1−α₁)(f/2)³  Equation 8

is solved for the e (sequencing error rate), alpha (proportion of case 1points), and f (fetal fraction). Where the Fi are defined as in Equation4 above. A closed form solution for fetal fraction is chosen to be thereal solution of

$\begin{matrix}{F \approx \frac{{\left( {{F\; 1} - 1} \right)F\; 2} \pm {\sqrt{F\; 2}\sqrt{{4F\; 1^{3}} + {F\; 2} - {3F\; 1\left( {2 + {F\; 1}} \right)F\; 2} + {4F\; 2^{2}}}}}{2\left( {{F\; 1^{2}} - {F\; 2}} \right)}} & {{Equation}\mspace{14mu} 9}\end{matrix}$

that is between 0 and 1.

To gauge the performance of estimators a simulated data-set ofHardy-Weinberg Equilibrium points (ai,di) was constructed with fetalfraction designed to be {1%, 3%, 5%, 10%, 15%, 20%, and 25%} and aconstant sequencing error rate of 1%. The 1% error rate is the currentlyaccepted rate for the sequencing machines and protocols we are using andis consistent with the graph of Illumina Genome analyzer II data shownin FIG. 3 above. Equation 9 was applied to the data and found, with theexception of a four point bias upwards, general agreement with the“known” fetal fraction. Interestingly, the sequencing error rate, e, isestimated to be just above 1%.

In the next mixture model example, thresholding or other filteringtechnique is again employed to remove data for polymorphisms fallinginto cases 3 and 4. However in this case, the sequencing error is known.This simplifies the resulting expression for fetal DNA fraction, f, asshown in equations 10. FIG. 8 shows that this version of a mixture modelprovided improved results compared to the approach employed withequation 9.

A similar approach is shown in equations 11 and 12. This approachrecognizes that only some sequencing errors add the minor allele count.Rather only one in every four sequencing errors should increase theminor allele count. FIG. 9 shows remarkably good agreement between theactual and estimated fetal fractions using this technique.

Model Cases (1+2), Sequencing Error Known

Since the sequencing error rate of the machines used is known to a greatextent, the bias and complexity of calculations can be reduced byeliminating e as a variable to be solved. Thus we obtain the system ofequations

F ₁=α₁ e+(1−α₁)(f/2)

F ₂=α₁ e ²+(1−α₁)(f/2)²  Equation 10

for fetal fraction f to obtain the solution:

$F \approx \frac{2\left( {{{eF}\; 1} - {F\; 2}} \right)}{\left( {e - {F\; 1}} \right)}$

FIG. 8 shows that using the machine error rate as a known parameterreduces the upward bias by a point.

Model Cases (1+2), Sequencing Error Known, Improved Error Models

To ameliorate bias in the model we expanded the error model of the aboveequations to account for the fact that not every sequencing error eventwill add to minor allele count A=ai in heterozygosity case 1.Furthermore, we allow for the fact that sequencing error events maycontribute to heterozygosity case 2 counts. Hence we determine fetalfraction F by solving for the following system of factorial momentrelations:

$\begin{matrix}{{F_{1} = {{\alpha_{1}e\text{/}4} + {\left( {1 - \alpha_{1}} \right)\left( {e + {f\text{/}2}} \right)}}}{F_{2} = {{\alpha_{1}\left( \frac{e}{4} \right)}^{2} + {\left( {1 - \alpha_{1}} \right)\left( {e + {f\text{/}2}} \right)^{2}}}}} & {{Equation}\mspace{14mu} 11}\end{matrix}$

which yields the solution

$\begin{matrix}{F \approx {\frac{{- 2}\left( {e^{2} - {5{eF}\; 1} + {4F\; 2}} \right)}{\left( {e - {4F\; 1}} \right)}.}} & {{Equation}\mspace{14mu} 12}\end{matrix}$

In FIG. 9 shows that simulated data using the machine error rate as aknown parameter, enhancing the case 1 and 2 error models greatly reducesthe upward bias to less than a point for fetal fraction below 0.2.

Implementation Options

Samples

Samples that are used in embodiments disclosed herein comprise genomicDNA that is cellular or cell-free. Cellular DNA is derived from wholecells by manually or mechanically extracting the genomic DNA from wholecells of the same or of differing genetic compositions. Cellular DNA canbe derived for example, from whole cells of the same genetic compositionderived from one subject, from a mixture of whole cells of differentsubjects, or from a mixture of whole cells that differ in geneticcomposition that are derived from one subject. Methods for extractinggenomic DNA from whole cells are known in the art, and differ dependingupon the nature of the source.

In some instances, it can be advantageous to fragment the cellulargenomic DNA. Fragmentation can be random, or it can be specific, asachieved, for example, using restriction endonuclease digestion. Methodsfor random fragmentation are well known in the art, and include, forexample, limited DNAse digestion, alkali treatment and physicalshearing. In certain embodiments, the sample nucleic acids are subjectedto fragmentation into fragments of approximately 500 or more base pairs,and to which next generation sequencing (NGS) methods can be readilyapplied. In one embodiment, sample nucleic acids are obtained from ascfDNA, which is not subjected to fragmentation.

Cell-free DNA is genomic DNA that naturally occurs as a mixture ofgenomic fragments typically found in biological fluids e.g. blood, of asubject. The genomic mixture can be derived from cells that naturallyrupture to release their genomic content by biological processes e.g.,apoptosis. A sample of cfDNA can comprise cfDNA derived from a mixtureof cells of different subjects of the same species, from a mixture ofcells from one subject that differ in genetic composition, or from amixture of cells from different species e.g. a subject.

Cell-free nucleic acids, including cell-free DNA, can be obtained byvarious methods known in the art from biological samples including butnot limited to plasma, serum and urine (Fan et al., Proc Natl Acad Sci105:16266-16271[2008]; Koide et al., Prenatal Diagnosis25:604-607[2005]; Chen et al., Nature Med. 2: 1033-1035 [1996]; Lo etal., Lancet 350: 485-487 [1997]; Botezatu et al., Clin Chem. 46:1078-1084, 2000; and Su et al., J Mol. Diagn. 6: 101-107 [2004]). Toseparate cfDNA from cells, fractionation, centrifugation (e.g., densitygradient centrifugation), DNA-specific precipitation, or high-throughputcell sorting and/or separation methods can be used. Commerciallyavailable kits for manual and automated separation of cfDNA areavailable (Roche Diagnostics, Indianapolis, Ind., Qiagen, Valencia,Calif., Macherey-Nagel, Duren, Del.).

The sample comprising the mixture of nucleic acids to which the methodsdescribed herein are applied may be a biological sample such as a tissuesample, a biological fluid sample, or a cell sample. In someembodiments, the mixture of nucleic acids is purified or isolated fromthe biological sample by any one of the known methods. A sample can be apurified or isolated polynucleotide. A biological fluid includes, asnon-limiting examples, blood, plasma, serum, sweat, tears, sputum,urine, sputum, ear flow, lymph, saliva, cerebrospinal fluid, ravages,bone marrow suspension, vaginal flow, transcervical lavage, brain fluid,ascites, milk, secretions of the respiratory, intestinal andgenitourinary tracts, amniotic fluid and leukophoresis samples. In someembodiments, the sample is a sample that is easily obtainable bynon-invasive procedures e.g. blood, plasma, serum, sweat, tears, sputum,urine, sputum, ear flow, saliva or feces. Preferably, the biologicalsample is a peripheral blood sample, or the plasma and serum fractions.In other embodiments, the biological sample is a swab or smear, a biopsyspecimen, or a cell culture. In another embodiment, the sample is amixture of two or more biological samples e.g. a biological sample cancomprise two or more of a biological fluid sample, a tissue sample, anda cell culture sample. As used herein, the terms “blood,” “plasma” and“serum” expressly encompass fractions or processed portions thereof.Similarly, where a sample is taken from a biopsy, swab, smear, etc., the“sample” expressly encompasses a processed fraction or portion derivedfrom the biopsy, swab, smear, etc.

In some embodiments, samples can be obtained from sources, including,but not limited to, samples from different individuals, differentdevelopmental stages of the same or different individuals, differentdiseased individuals (e.g., individuals with cancer or suspected ofhaving a genetic disorder), normal individuals, samples obtained atdifferent stages of a disease in an individual, samples obtained from anindividual subjected to different treatments for a disease, samples fromindividuals subjected to different environmental factors, or individualswith predisposition to a pathology, or individuals with exposure to aninfectious disease agent (e.g., HIV).

In one embodiment, the sample is a maternal sample that is obtained froma pregnant female, for example a pregnant woman. In this instance, thesample can be analyzed using the methods described herein to provide aprenatal diagnosis of potential chromosomal abnormalities in the fetus.The maternal sample can be a tissue sample, a biological fluid sample,or a cell sample. A biological fluid includes, as non-limiting examples,blood, plasma, serum, sweat, tears, sputum, urine, sputum, ear flow,lymph, saliva, cerebrospinal fluid, ravages, bone marrow suspension,vaginal flow, transcervical lavage, brain fluid, ascites, milk,secretions of the respiratory, intestinal and genitourinary tracts, andleukophoresis samples. In another embodiment, the maternal sample is amixture of two or more biological samples e.g. a biological sample cancomprise two or more of a biological fluid sample, a tissue sample, anda cell culture sample. In some embodiments, the sample is a sample thatis easily obtainable by non-invasive procedures e.g. blood, plasma,serum, sweat, tears, sputum, urine, sputum, ear flow, saliva and feces.In some embodiments, the biological sample is a peripheral blood sample,or the plasma and serum fractions. In other embodiments, the biologicalsample is a swab or smear, a biopsy specimen, or a cell culture.

Samples can also be obtained from in vitro cultured tissues, cells, orother polynucleotide-containing sources. The cultured samples can betaken from sources including, but not limited to, cultures (e.g., tissueor cells) maintained in different media and conditions (e.g., pH,pressure, or temperature), cultures (e.g., tissue or cells) maintainedfor different periods of length, cultures (e.g., tissue or cells)treated with different factors or reagents (e.g., a drug candidate, or amodulator), or cultures of different types of tissue or cells. Methodsof isolating nucleic acids from biological sources are well known andwill differ depending upon the nature of the source as explained above.

Polymorphisms for Use in Identifying Genomic Fraction

As explained, polymorphisms may be used to assess the fetal fraction.The allele fraction and zygosity of one or more polymorphisms is used inthe assessment. Examples of useful polymorphisms include, withoutlimitation, single nucleotide polymorphisms (SNPs), tandem SNPs,small-scale multi-base deletions or insertions, called IN-DELS (alsocalled deletion insertion polymorphisms or DIPs), Multi-NucleotidePolymorphisms (MNPs), Short Tandem Repeats (STRs), restriction fragmentlength polymorphisms (RFLPs), deletions, including microdeletions,insertions, including microinsertions, duplications, inversions,translocations, multiplications, complex multi-site variants, copynumber variations (CNVs), and polymorphisms comprising any other changeof sequence in a chromosome.

In some embodiments, polymorphisms that are used in the disclosedmethods include SNPs and/or STRs. SNP polymorphisms can be single SNP,tandem SNPs. Single SNPs include individual SNPs, and tag SNPs i.e. SNPspresent in a haplotype, and/or a haplotype block. In some embodiments,combinations of polymorphisms are used. For example, differences in copynumber can be detected by comparison of a combination of polymorphicsequences comprising one or more SNPs and one or more STRs.

In general, any polymorphic site that can be encompassed by the readsgenerated by the sequencing methods described herein can be used toidentify genomic fraction in samples comprising DNA of differentgenomes. Polymorphic sequences useful for practicing the methods of theinvention are available from a variety of publicly accessible databases,which are continuously expanding. For example, useful databases includewithout limitation Human SNP Database at world wide web addresswi.mit.edu, NCBI dbSNP Home Page at world wide web addressncbi.nlm.nih.gov, world wide web address lifesciences.perkinelmer.com,Celera Human SNP database at world wide web address celera.com, the SNPDatabase of the Genome Analysis Group (GAN) at world wide web addressgan.iarc.fr, ATCC short tandem repeat (STR) database at world wide webaddress atcc.org, and the HapMap database at world wide web addresshapmap.org.

The number of polymorphisms that can be used in a fetal fractionassessment can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000 or more. For example, it is estimated that thehuman genome comprises at least about 10 million SNPs. Therefore, thenumber of available polymorphisms that can be genotyped in a sample froma human subject can be at least about 10 million SNPs, as well as manyother types of polymorphisms that are present in any one human genome.In some embodiments, identification of one or more polymorphisms in afirst genome of a sample comprising a mixture of DNA e.g. cfDNA, of afirst and a second genome is performed by whole genome sequencing usinga NGS method as described herein. In some embodiments, the whole genomesequencing method is an NGS method that identifies the polymorphicsequences by massively parallel sequencing clonally amplified nucleicacid molecules or by massively parallel sequencing of single nucleicacid molecules i.e. single molecule sequencing.

Applications

The fraction of nucleic acid originating from each of two distinctgenomic sources in a sample can be used for various purposes. In variousembodiments described herein, the fraction of fetal DNA in cell free DNAof a material sample is used to facilitate prenatal diagnoses and toassist in making decisions concerning treatment of pregnancies. In otherembodiments, the genomes under consideration are not maternal and fetal.Various examples of genomic sources for determining fractional genomepresence are presented below.

Cell-free fetal DNA and RNA circulating in maternal blood can be usedfor the early non-invasive prenatal diagnosis (NIPD) of an increasingnumber of genetic conditions, both for pregnancy management and to aidreproductive decision-making. Small amounts of circulating fetal DNA arepresent in the maternal bloodstream during pregnancy (Lo et al., Lancet350:485-487[1997]). Thought to originate from dying placental cells,cell-free fetal DNA has been shown to consists of short fragmentstypically fewer than 200 bp in length Chan et al., Clin Chem50:88-92[2004]), which can be discerned as early as 4 weeks gestation(Illanes et al., Early Human Dev 83:563-566[2007]), and known to becleared from the maternal circulation within hours of delivery (Lo etal., Am J Hum Genet 64:218-224 [1999]). In addition to cfDNA, fragmentsof cell-free fetal RNA (cfRNA) can also be discerned in the maternalbloodstream, originating from genes that are transcribed in the fetus orplacenta. The extraction and subsequent analysis of these fetal geneticelements from a maternal blood sample offers novel opportunities forNIPD.

As explained, the disclosed methods determine the fraction of a secondgenome in a biological sample. The methods optionally determine thepresence or absence of a number of disorders in a blood samplecomprising a mixture of DNA (such as cfDNA) of a first and a secondgenome. In some embodiments, determination the fetal fraction maycomprise (a) genome sequencing at least a portion of the mixture ofcfDNA to obtain a plurality of sequence tags; (b) determining in theplurality of sequence tags the presence or absence of multiplepolymorphisms, and (c) associating the multiple polymorphisms with thefirst and/or second genome in the mixture. In preferred embodiments, themixture is unenriched for the multiple polymorphisms. Identification ofthe multiple polymorphisms in the mixture of DNA is performed bycomparing the sequence of the mapped tags obtained by the whole genomesequencing method to multiple reference polymorphisms, as describedherein.

In embodiment disclosed above, the first genome is a fetal genome, and asecond genome is a maternal genome. In another embodiment, the firstgenome is a genome of an unaffected cell and the second genome is agenome from an affected cell e.g. a cancer cell. In some embodiments,the affected and unaffected cells are derived from the same subject. Forexample, the affected cell can be a cell whose genome has been alteredby a disorder. In some embodiments the disorder is a monogenic disorder.In other embodiments, the disorder is a polygenic disorder. Disorderscan be identified by a single polymorphism e.g. a tag SNP, or bymultiple polymorphisms present in a haplotype. In some embodiments, themultiple polymorphisms identified according to the present method arepresent in a haplotype block.

The disorders that can be identified with the aid of the present methodare genetic disorders, which are illnesses caused at least in part byabnormalities in genes or chromosomes. Knowledge of a fetal fraction ina sample can assist in identifying such disorders in a prenatal context.Disorders identified by the present method include monogenic i.e. singlegene, disorders and polygenic i.e. complex disorders. Single genedisorders include autosomal dominant, autosomal recessive, X-linkeddominant, X-linked recessive, and Y-linked.

In autosomal dominant disorders, only one mutated copy of the gene willbe necessary for a person to be affected by the disorder. Typically, anaffected subject has one affected parent, and there is a 50% chance thatthe offspring will inherit the mutated gene. Conditions that areautosomal dominant sometimes have reduced penetrance, which means thatalthough only one mutated copy is needed, not all individuals whoinherit that mutation go on to develop the disease. Examples ofautosomal dominant disorders that can be identified by the presentmethod include without limitation familial hypercholesterolemia,hereditary spherocytosis, Marfan syndrome, neurofibromatosis type 1,hereditary nonpolyposis colorectal cancer, and hereditary multipleexostoses, and Huntington disease.

Autosomal recessive disorders detected using the present method includeSickle cell anemia, Cystic fibrosis, Tay-Sachs disease, Tay-Sachsdisease, Mucopolysaccharidoses, Glycogen storage diseases, andGalactosemia. X-linked disorders detected by the present method includeDuchenne muscular dystrophy and hemophilia. In autosomal recessivedisorders, two copies of the gene must be mutated for a subject to beaffected by an autosomal recessive disorder. An affected subject usuallyhas unaffected parents who each carry a single copy of the mutated gene(and are referred to as carriers). Two unaffected people who each carryone copy of the mutated gene have a 25% chance with each pregnancy ofhaving a child affected by the disorder. Examples of this type ofdisorder that can be identified by the present method include are cysticfibrosis, sickle-cell disease, Tay-Sachs disease, Niemann-Pick disease,spinal muscular atrophy, and Roberts syndrome. Certain other phenotypes,such as wet versus dry earwax, are also determined in an autosomalrecessive fashion. X-linked dominant disorders are caused by mutationsin genes on the X chromosome. Only a few disorders have this inheritancepattern, with a prime example being X-linked hypophosphatemic rickets.Males and females are both affected in these disorders, with malestypically being more severely affected than females. Some X-linkeddominant conditions such as Rett syndrome, incontinentiapigmenti type 2and Aicardi syndrome are usually fatal in males, and are thereforepredominantly seen in females. Exceptions to this finding are extremelyrare cases in which boys with Klinefelter syndrome (47,XXY) also inheritan X-linked dominant condition and exhibit symptoms more similar tothose of a female in terms of disease severity. The chance of passing onan X-linked dominant disorder differs between men and women. The sons ofa man with an X-linked dominant disorder will all be unaffected (sincethey receive their father's Y chromosome), and his daughters will allinherit the condition. A woman with an X-linked dominant disorder has a50% chance of having an affected fetus with each pregnancy, although itshould be noted that in cases such as incontinentiapigmenti only femaleoffspring are generally viable. In addition, although these conditionsdo not alter fertility per se, individuals with Rett syndrome or Aicardisyndrome rarely reproduce.

The present method can also facilitate identification of polymorphismsassociated with X-linked disorders. X-linked recessive conditions arealso caused by mutations in genes on the X chromosome. Males are morefrequently affected than females, and the chance of passing on thedisorder differs between men and women. The sons of a man with anX-linked recessive disorder will not be affected, and his daughters willcarry one copy of the mutated gene. A woman who is a carrier of anX-linked recessive disorder (X^(R)X^(r)) has a 50% chance of having sonswho are affected and a 50% chance of having daughters who carry one copyof the mutated gene and are therefore carriers. X-linked recessiveconditions include without limitation the serious diseases Hemophilia A,Duchenne muscular dystrophy, and Lesch-Nyhan syndrome as well as commonand less serious conditions such as male pattern baldness and red-greencolor blindness. X-linked recessive conditions can sometimes manifest infemales due to skewed X-inactivation or monosomy X (Turner syndrome).

Y-linked disorders are caused by mutations on the Y chromosome. Becausemales inherit a Y chromosome from their fathers, every son of anaffected father will be affected. Because females inherit an Xchromosome from their fathers, female offspring of affected fathers arenever affected. Since the Y chromosome is relatively small and containsvery few genes, there are relatively few Y-linked disorders. Often thesymptoms include infertility, which may be circumvented with the help ofsome fertility treatments. Examples are male infertility andhypertrichosis pinnae.

As explained the disclosed methods for detecting genomic fractions in asample can be used to facilitate detection of aneuploidy from materialsamples. In some embodiments, the aneuploidy is a complete chromosomaltrisomy or monosomy, or a partial trisomy or monosomy. Partialaneuploidies are caused by loss or gain of part of a chromosome, andencompass chromosomal imbalances resulting from unbalancedtranslocations, unbalanced inversions, deletions and insertions. By far,the most common known aneuploidy compatible with life is trisomy 21 i.e.Down Syndrome (DS), which is caused by the presence of part or all ofchromosome 21. Rarely, DS can be cause by an inherited or sporadicdefect whereby an extra copy of all or part of chromosome 21 becomesattached to another chromosome (usually chromosome 14) to form a singleaberrant chromosome. DS is associated with intellectual impairment,severe learning difficulties and excess mortality caused by long-termhealth problems such as heart disease. Other aneuploidies with knownclinical significance include Edward syndrome (trisomy 18) and PatauSyndrome (trisomy 13), which are frequently fatal within the first fewmonths of life. Abnormalities associated with the number of sexchromosomes are also known and include monosomy X e.g. Turner syndrome(XO), and triple X syndrome (XXX) in female births and Kleinefeltersyndrome (XXY) and XYY syndrome in male births, which are all associatedwith various phenotypes including sterility and reduction inintellectual skills. Monosomy X [45,X] is a common cause of earlypregnancy loss accounting for about 7% of spontaneous abortions. Basedon the liveborn frequency of 45,X (also called Turner syndrome) of1-2/10,000, it is estimated that less than 1% of 45,X conceptuses willsurvive to term. About 30% of Turners syndrome patients are mosaic withboth a 45,X cell line and either a 46,XX cell line or one containing arearranged X chromosome (Hook and Warburton 1983). The phenotype in aliveborn infant is relatively mild considering the high embryoniclethality and it has been hypothesized that possibly all livebornfemales with Turner syndrome carry a cell line containing two sexchromosomes. Monosomy X can occur in females as 45,X or as 45,X/46XX,and in males as 45,X/46XY. Autosomal monosomies in human are generallysuggested to be incompatible with life; however, there is quite a numberof cytogenetic reports describing full monosomy of one chromosome 21 inlive born children (Vosranovalet al., Molecular Cytogen. 1:13[2008];Joosten et al., Prenatal Diagn. 17:271-5[1997]. The method of theinvention can be used to diagnose these and other chromosomalabnormalities prenatally.

According to some embodiments, fetal fraction can useful in determiningthe presence or absence of chromosomal trisomies of any one ofchromosomes 1-22, X and Y. Examples of chromosomal trisomies that can bedetected according to the present method include without limitationtrisomy 21 (T21; Down Syndrome), trisomy 18 (T18; Edward's Syndrome),trisomy 16 (T16), trisomy 20 (T20), trisomy 22 (T22; Cat Eye Syndrome),trisomy 15 (T15; PraderWilli Syndrome), trisomy 13 (T13; PatauSyndrome), trisomy 8 (T8; Warkany Syndrome), trisomy 9, and the XXY(Kleinefelter Syndrome), XYY, or XXX trisomies. Complete trisomies ofother autosomes existing in a non-mosaic state are lethal, but can becompatible with life when present in a mosaic state. It will beappreciated that various complete trisomies, whether existing in amosaic or non-mosaic state, and partial trisomies can be determined infetal cfDNA according to the teachings of the present invention.

Non-limiting examples of partial trisomies that can be determined by thepresent method include, but are not limited to, partial trisomy 1q32-44,trisomy 9 p, trisomy 4 mosaicism, trisomy 17p, partial trisomy4q26-qter, partial 2p trisomy, partial trisomy 1q, and/or partialtrisomy 6p/monosomy 6q.

The methods disclosed herein can also be used to help determinechromosomal monosomy X, chromosomal monosomy 21, and partial monosomiessuch as, monosomy 13, monosomy 15, monosomy 16, monosomy 21, andmonosomy 22, which are known to be involved in pregnancy miscarriage.Partial monosomy of chromosomes typically involved in completeaneuploidy can also be determined by the method of the invention.Non-limiting examples of deletion syndromes that can be determinedaccording to the present method include syndromes caused by partialdeletions of chromosomes. Examples of partial deletions that can bedetermined according to the method of the invention include withoutlimitation partial deletions of chromosomes 1, 4, 5, 7, 11, 18, 15, 13,17, 22 and 10, which are described in the following.

1q21.1 deletion syndrome or 1q21.1 (recurrent) microdeletion is a rareaberration of chromosome 1. Next to the deletion syndrome, there is alsoa 1q21.1 duplication syndrome. While there is a part of the DNA missingwith the deletion syndrome on a particular spot, there are two or threecopies of a similar part of the DNA on the same spot with theduplication syndrome. Literature refers to both the deletion and theduplication as the 1q21.1 copy-number variations (CNV). The 1q21.1deletion can be associated with the TAR Syndrome (Thrombocytopenia withAbsent radius).

Wolf-Hirschhorn syndrome (WHS) (OMIN #194190) is a contiguous genedeletion syndrome associated with a hemizygous deletion of chromosome4p16.3. Wolf-Hirschhorn syndrome is a congenital malformation syndromecharacterized by pre- and postnatal growth deficiency, developmentaldisability of variable degree, characteristic craniofacial features(‘Greek warrior helmet’ appearance of the nose, high forehead, prominentglabella, hypertelorism, high-arched eyebrows, protruding eyes,epicanthal folds, short philtrum, distinct mouth with downturnedcorners, and micrognathia), and a seizure disorder.

Partial deletion of chromosome 5, also known as 5p- or 5p minus, andnamed Cris du Chat syndrome (OMIN#123450), is caused by a deletion ofthe short arm (p arm) of chromosome 5 (5p15.3-p15.2). Infants with thiscondition often have a high-pitched cry that sounds like that of a cat.The disorder is characterized by intellectual disability and delayeddevelopment, small head size (microcephaly), low birth weight, and weakmuscle tone (hypotonia) in infancy, distinctive facial features andpossibly heart defects.

Williams-Beuren Syndrome also known as chromosome 7q11.23 deletionsyndrome (OMIN 194050) is a contiguous gene deletion syndromeresultingin a multisystem disorder caused by hemizygous deletion of 1.5 to 1.8 Mbon chromosome 7q11.23, which contains approximately 28 genes.

Jacobsen Syndrome, also known as 11q deletion disorder, is a rarecongenital disorder resulting from deletion of a terminal region ofchromosome 11 that includes band 11q24.1. It can cause intellectualdisabilities, a distinctive facial appearance, and a variety of physicalproblems including heart defects and a bleeding disorder.

Partial monosomy of chromosome 18, known as monosomy 18p is a rarechromosomal disorder in which all or part of the short arm (p) ofchromosome 18 is deleted (monosomic). The disorder is typicallycharacterized by short stature, variable degrees of mental retardation,speech delays, malformations of the skull and facial (craniofacial)region, and/or additional physical abnormalities. Associatedcraniofacial defects may vary greatly in range and severity from case tocase.

Conditions caused by changes in the structure or number of copies ofchromosome 15 include Angelman Syndrome and Prader-Willi Syndrome, whichinvolve a loss of gene activity in the same part of chromosome 15, the15q11-q13 region. It will be appreciated that several translocations andmicrodeletions can be asymptomatic in the carrier parent, yet can causea major genetic disease in the offspring. For example, a healthy motherwho carries the 15q11-q13 microdeletion can give birth to a child withAngelman syndrome, a severe neurodegenerative disorder. Thus, thepresent invention can be used to identify such a partial deletion andother deletions in the fetus.

Partial monosomy 13q is a rare chromosomal disorder that results when apiece of the long arm (q) of chromosome 13 is missing (monosomic).Infants born with partial monosomy 13q may exhibit low birth weight,malformations of the head and face (craniofacial region), skeletalabnormalities (especially of the hands and feet), and other physicalabnormalities. Mental retardation is characteristic of this condition.The mortality rate during infancy is high among individuals born withthis disorder. Almost all cases of partial monosomy 13q occur randomlyfor no apparent reason (sporadic).

Smith-Magenis syndrome (SMS—OMIM #182290) is caused by a deletion, orloss of genetic material, on one copy of chromosome 17. This well-knownsyndrome is associated with developmental delay, mental retardation,congenital anomalies such as heart and kidney defects, andneurobehavioral abnormalities such as severe sleep disturbances andself-injurious behavior. Smith-Magenis syndrome (SMS) is caused in mostcases (90%) by a 3.7-Mb interstitial deletion in chromosome 17p11.2.

22q11.2 deletion syndrome, also known as DiGeorge syndrome, is asyndrome caused by the deletion of a small piece of chromosome 22. Thedeletion (22 q11.2) occurs near the middle of the chromosome on the longarm of one of the pair of chromosome. The features of this syndrome varywidely, even among members of the same family, and affect many parts ofthe body. Characteristic signs and symptoms may include birth defectssuch as congenital heart disease, defects in the palate, most commonlyrelated to neuromuscular problems with closure (velo-pharyngealinsufficiency), learning disabilities, mild differences in facialfeatures, and recurrent infections. Microdeletions in chromosomal region22q11.2 are associated with a 20 to 30-fold increased risk ofschizophrenia.

Deletions on the short arm of chromosome 10 are associated with aDiGeorge Syndrome like phenotype. Partial monosomy of chromosome 10p israre but has been observed in a portion of patients showing features ofthe DiGeorge Syndrome.

In one embodiment, the method of the invention is used to determinepartial monosomies including but not limited to partial monosomy ofchromosomes 1, 4, 5, 7, 11, 18, 15, 13, 17, 22 and 10, e.g. partialmonosomy 1q21.11, partial monosomy 4p16.3, partial monosomy5p15.3-p15.2, partial monosomy 7q11.23, partial monosomy 11q24.1,partial monosomy 18p, partial monosomy of chromosome 15 (15q11-q13),partial monosomy 13q, partial monosomy 17p11.2, partial monosomy ofchromosome 22 (22q11.2), and partial monosomy 10p can also be determinedusing the method.

Other partial monosomies that can be determined according to the methodof the invention include unbalanced translocation t(8;11)(p23.2;p15.5);11q23 microdeletion; 17p11.2 deletion; 22q13.3 deletion; Xp22.3microdeletion; 10p14 deletion; 20p microdeletion,[del(22)(q11.2q11.23)], 7q11.23 and 7q36 deletions; 1p36 deletion; 2pmicrodeletion; neurofibromatosis type 1 (17q11.2 microdeletion), Yqdeletion; 4p16.3 microdeletion; 1p36.2 microdeletion; 11q14 deletion;19q13.2 microdeletion; Rubinstein-Taybi (16 p13.3 microdeletion); 7p21microdeletion; Miller-Dieker syndrome (17p13.3); and 2q37 microdeletion.Partial deletions can be small deletions of part of a chromosome, orthey can be microdeletions of a chromosome where the deletion of asingle gene can occur.

Several duplication syndromes caused by the duplication of part ofchromosome arms have been identified (see OMIN [Online MendelianInheritance in Man viewed online at ncbi.nlm.nih.gov/omim]) In oneembodiment, the present method can be used to determine the presence orabsence of duplications and/or multiplications of segments of any one ofchromosomes 1-22, X and Y. Non-limiting examples of duplicationssyndromes that can be determined according to the present method includeduplications of part of chromosomes 8, 15, 12, and 17, which aredescribed in the following.

8p23.1 duplication syndrome is a rare genetic disorder caused by aduplication of a region from human chromosome 8. This duplicationsyndrome has an estimated prevalence of 1 in 64,000 births and is thereciprocal of the 8p23.1 deletion syndrome. The 8p23.1 duplication isassociated with a variable phenotype including one or more of speechdelay, developmental delay, mild dysmorphism, with prominent foreheadand arched eyebrows, and congenital heart disease (CHD).

Chromosome 15q Duplication Syndrome (Dup15q) is a clinicallyidentifiable syndrome which results from duplications of chromosome15q11-13.1 Babies with Dup15q usually have hypotonia (poor muscle tone),growth retardation; they may be born with a cleft lip and/or palate ormalformations of the heart, kidneys or other organs; they show somedegree of cognitive delay/disability (mental retardation), speech andlanguage delays, and sensory processing disorders.

Pallister Killian syndrome is a result of extra #12 chromosome material.There is usually a mixture of cells (mosaicism), some with extra #12material, and some that are normal (46 chromosomes without the extra #12material). Babies with this syndrome have many problems including severemental retardation, poor muscle tone, “coarse” facial features, and aprominent forehead. They tend to have a very thin upper lip with athicker lower lip and a short nose. Other health problems includeseizures, poor feeding, stiff joints, cataracts in adulthood, hearingloss, and heart defects. Persons with Pallister Killian have a shortenedlifespan.

Individuals with the genetic condition designated as dup(17)(p11.2p11.2)or dup 17p carry extra genetic information (known as a duplication) onthe short arm of chromosome 17. Duplication of chromosome 17p11.2underlies Potocki-Lupski syndrome (PTLS), which is a newly recognizedgenetic condition with only a few dozen cases reported in the medicalliterature. Patients who have this duplication often have low muscletone, poor feeding, and failure to thrive during infancy, and alsopresent with delayed development of motor and verbal milestones. Manyindividuals who have PTLS have difficulty with articulation and languageprocessing. In addition, patients may have behavioral characteristicssimilar to those seen in persons with autism or autism-spectrumdisorders. Individuals with PTLS may have heart defects and sleep apnea.A duplication of a large region in chromosome 17p12 that includes thegene PMP22 is known to cause Charcot-Marie Tooth disease.

CNV have been associated with stillbirths. However, due to inherentlimitations of conventional cytogenetics, the contribution of CNV tostillbirth is thought to be underrepresented (Harris et al., PrenatalDiagn 31:932-944[2011]). The present methods are useful in assistingdetermination of the presence of partial aneuploidies e.g. deletions andmultiplications of chromosome segments, and can be used to help identifyand determine the presence or absence of CNV that are associated withstillbirths.

The present method can also assist in identifying polymorphismsassociated with genetic disorders that are complex, multifactorial, orpolygenic, meaning that they are likely associated with the effects ofmultiple genes in combination with lifestyle and environmental factors.Multifactorial disorders include for example, heart disease anddiabetes. Although complex disorders often cluster in families, they donot have a clear-cut pattern of inheritance. On a pedigree, polygenicdiseases do tend to “run in families”, but the inheritance does is notsimple as is with Mendelian diseases. Strong environmental componentsare associated with many complex disorders e.g., blood pressure. Thepresent method can be used to identify polymorphisms that are associatedwith polygenic disorders including but not limited to asthma, autoimmunediseases such as multiple sclerosis, cancers, ciliopathies, cleftpalate, diabetes, heart disease, hypertension, inflammatory boweldisease, mental retardation, mood disorder, obesity, refractive error,and infertility. In some embodiments, the polymorphisms are SNPs. Inother embodiments, the polymorphisms are STRs. In yet other embodiments,the polymorphisms are a combination of SNPs and STRs.

In one embodiment, identification of the polymorphic sequencesassociated with disorders comprises sequencing at least a portion of thecellular genome corresponding to the second genome in the mixture ofcfDNA. Identification of polymorphic sequences contributed by a firstgenome is performed by determining the sequence at multiple polymorphicsites in a first sample containing DNA molecules derived essentiallyfrom only a second genome, determining the sequence at the correspondingmultiple polymorphic sites in a second sample containing a mixture ofDNA molecules derived from a first and a second genome, and comparingthe polymorphic sequences determined in both samples thereby identifyingmultiple polymorphisms in a first genome of a sample comprising amixture of two genomes. For example, identification of polymorphicsequences contributed by a fetal genome i.e. first genome, is performedby determining the sequence at multiple polymorphic sites in a maternalbuffy coat sample i.e. a sample containing DNA molecules derivedessentially from only a second genome, determining the sequence at thecorresponding multiple polymorphic sites in a purified plasma samplei.e. a second sample containing a mixture of cfDNA molecules derivedfrom the fetal and the maternal genomes, and comparing the polymorphicsequences determined in both samples to identify multiple fetalpolymorphisms. In one embodiment, the first genome is a fetal genome,and a second genome is a maternal genome. In another embodiment, thefirst genome is a genome of an unaffected cell and the second genome isa genome from an affected cell. In some embodiments, the affected andunaffected cells are derived from the same subject. For example, theaffected cell can be a cell whose genome has been altered by a disorder.

In one embodiment, the disclosed methods of estimating genomic fractionassist in detecting cancer in a patient. In various examples, a canceris detected by a method comprising: providing a sample from a patientcomprising a mixture of genomes derived from normal i.e. unaffected, andcancerous i.e. affected, cells; and identifying multiple polymorphismsassociated with cancer. In some embodiments, the sample is selected fromblood, plasma, serum and urine. In some embodiments, the sample is aplasma sample. In other embodiments, the sample is a urine sample.

In one embodiment, identifying multiple polymorphisms associated withcancer comprises enriching the DNA in the sample for polymorphic targetsequences. In other embodiments, enrichment of the sample forpolymorphic target sequences is not performed. In some embodiments,identifying multiple polymorphisms associated with cancer comprisesquantifying the number of copies of the polymorphic sequence.

Cancers that can be identified and/or monitored according to the methodof the invention include solid tumors, as well as, hematologic tumorsand/or malignancies. Various cancers to be treated include sarcomas,carcinomas, and adenocarcinomas not limited to breast cancer, lungcancer, colorectal cancer, pancreatic cancer, ovarian cancer, prostatecancer, renal carcinoma, hepatoma, brain cancer, melanoma, multiplemyeloma, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, childhoodlymphomas, and lymphomas of lymphocytic and cutaneous origin, leukemia,childhood leukemia, hairy-cell leukemia, acute lymphocytic leukemia,acute myelocytic leukemia, chronic lymphocytic leukemia, chronicmyelocytic leukemia, chronic myelogenous leukemia, and mast cellleukemia, myeloid neoplasms, mast cell neoplasms, hematologic tumor, andlymphoid tumor, including metastatic lesions in other tissues or organsdistant from the primary tumor site.

The methods of the present invention are useful, for example, indiagnosing or determining a prognosis in a disease condition known to beassociated with a specific haplotype(s), to determine novel haplotypes,and to detect haplotype associations with responsiveness topharmaceuticals. The association of multiple polymorphic sequences withmultiple disorders can be determined from the identity of a singlepolymorphic sequence for each of the multiple disorders. Alternatively,association of multiple polymorphic sequences with multiple disorderscan be determined from the identity of multiple polymorphic sequencesfor each of the multiple disorders.

Conventional genotyping techniques have been limited to identifyingpolymorphisms in short genomic regions of a few kilobases, andidentification of haplotypes has relied on family data and statisticalestimation using computational algorithms. Whole genome sequencingenables the identification of haplotypes by directly identifying thepolymorphisms on a genome. The identification of the haplotypesaccording to various embodiments is not limited by the interveningdistance between polymorphisms. In some embodiments, a method compriseswhole genome sequencing maternal cellular DNA. Maternal cellular DNA canbe obtained from a biological sample devoid of fetal genomic DNA. Forexample, maternal DNA can be obtained from the buffy coat layer of amaternal blood. Haplotypes comprising a plurality of polymorphicsequences that span entire chromosomes can be determined. In oneembodiment, the fetal haplotypes are compared to knowndisorder-associated haplotypes, and based on a match of the fetalhaplotype with any one of the known disorder-associated haplotypesindicates that the fetus has the disorder or that the fetus issusceptible for the disorder. Fetal haplotypes can also be compared tohaplotypes associated with treatment responsiveness or unresponsivenessof the specific polymorphism. Comparison of the identified fetalhaplotypes to known haplotype databases allow for the diagnosis and/orprognosis of a disorder. Any biological sample comprising a mixture offetal and maternal cfDNA can be used to determine the presence orabsence of the fetal disorder. Preferably, the biological sample isselected from blood, or fractions thereof including plasma, or urine. Inone embodiment, the biological sample is a blood sample. In anotherembodiment, the biological sample is a plasma sample. In yet anotherembodiment, the biological sample is a urine sample.

In one embodiment, the invention provides a method for determining thepresence or absence of multiple fetal disorders, comprising (a)obtaining a maternal blood sample comprising a cell-free mixture offetal and maternal DNA, (b) whole genome sequencing at least a portionof the cell-free mixture of fetal and maternal DNA, thereby obtaining aplurality of sequence tags; (c) determining multiple fetal polymorphismsin the sequence tags, and (d) determining the presence or absence ofmultiple fetal disorders. Examples of multiple fetal disorders that canbe identified according to the present method include monogenic andpolygenic disorders described herein.

In one embodiment, the invention provides a method for determining thepresence or absence of multiple fetal disorders that comprisesidentifying multiple fetal polymorphisms associated with multipledisorders related haplotypes. In some embodiments, each of thehaplotypes comprises at least at least two, at least three, at leastfour, at least five, at least ten or at least fifteen different tagpolymorphisms. The tag polymorphisms present in the haplotype can be ofthe same type of polymorphism e.g. all tag SNP polymorphisms, or can bea combination of polymorphisms e.g. tag SNPs and tag deletions. In oneembodiment, the polymorphisms are tag SNPs. In another embodiment, thepolymorphisms are tag STRs. In yet another embodiment, the polymorphismsare a combination of tag SNPs and tag STRs. The tag polymorphisms can bein coding and/or non-coding regions of the genome. Identification of thepolymorphisms is performed by whole genome sequencing using NGStechnologies as described herein.

The invention provides a method for identifying copy number variations(CNV) as polymorphisms of a sequence of interest in a test sample thatcomprises a mixture of nucleic acids derived from two different genomes,and which are known or are suspected to differ in the amount of one ormore sequence of interest. Copy number variations determined by themethod of the invention include gains or losses of entire chromosomes,alterations involving very large chromosomal segments that aremicroscopically visible, and an abundance of sub-microscopic copy numbervariation of DNA segments ranging from kilobases (kb) to megabases (Mb)in size.

CNV in the human genome significantly influence human diversity andpredisposition to disease (Redon et al., Nature 23:444-454[2006],Shaikhet al. Genome Res 19:1682-1690[2009]). CNVs have been known tocontribute to genetic disease through different mechanisms, resulting ineither imbalance of gene dosage or gene disruption in most cases. Inaddition to their direct correlation with genetic disorders, CNVs areknown to mediate phenotypic changes that can be deleterious. Recently,several studies have reported an increased burden of rare or de novoCNVs in complex disorders such as Autism, ADHD, and schizophrenia ascompared to normal controls, highlighting the potential pathogenicity ofrare or unique CNVs (Sebat et al., 316:445-449[2007]; Walsh et al.,Science 320:539-543 [2008]). CNV arise from genomic rearrangements,primarily owing to deletion, duplication, insertion, and unbalancedtranslocation events.

Embodiments of the invention provide for a method to assess copy numbervariation of a sequence of interest e.g. a clinically-relevant sequence,in a test sample that comprises a mixture of nucleic acids derived fromtwo different genomes, and which are known or are suspected to differ inthe amount of one or more sequence of interest. The mixture of nucleicacids is derived from two or more types of cells. In one embodiment, themixture of nucleic acids is derived from normal and cancerous cellsderived from a subject suffering from a medical condition e.g. cancer.

It is believed that many solid tumors, such as breast cancer, progressfrom initiation to metastasis through the accumulation of severalgenetic aberrations. [Sato et al., Cancer Res., 50: 7184-7189 [1990];Jongsma et al., J Clin Pathol: Mol Path 55:305-309[2002])]. Such geneticaberrations, as they accumulate, may confer proliferative advantages,genetic instability and the attendant ability to evolve drug resistancerapidly, and enhanced angiogenesis, proteolysis and metastasis. Thegenetic aberrations may affect either recessive “tumor suppressor genes”or dominantly acting oncogenes. Deletions and recombination leading toloss of heterozygosity (LOH) are believed to play a major role in tumorprogression by uncovering mutated tumor suppressor alleles.

cfDNA has been found in the circulation of patients diagnosed withmalignancies including but not limited to lung cancer (Pathak et al.Clin Chem 52:1833-1842[2006]), prostate cancer (Schwartzenbach et al.Clin Cancer Res 15:1032-8[2009]), and breast cancer (Schwartzenbach etal. available online at breast-cancer-research.com/content/11/5/R71[2009]). Identification of genomic instabilities associated with cancersthat can be determined in the circulating cfDNA in cancer patients is apotential diagnostic and prognostic tool. In one embodiment, the methodof the invention assesses CNV of a sequence of interest in a samplecomprising a mixture of nucleic acids derived from a subject that issuspected or is known to have cancer e.g. carcinoma, sarcoma, lymphoma,leukemia, germ cell tumors and blastoma. In one embodiment, the sampleis a plasma sample derived (processes) from peripheral blood and thatcomprises a mixture of cfDNA derived from normal and cancerous cells. Inanother embodiment, the biological sample that is needed to determinewhether a CNV is present is derived from a mixture of cancerous andnon-cancerous cells from other biological fluids including but notlimited to serum, sweat, tears, sputum, urine, sputum, ear flow, lymph,saliva, cerebrospinal fluid, ravages, bone marrow suspension, vaginalflow, transcervical lavage, brain fluid, ascites, milk, secretions ofthe respiratory, intestinal and genitourinary tracts, and leukophoresissamples, or in tissue biopsies, swabs or smears.

The sequence of interest is a nucleic acid sequence that is known or issuspected to play a role in the development and/or progression of thecancer. Examples of a sequence of interest include nucleic acidssequences that are amplified or deleted in cancerous cells as describedin the following.

Dominantly acting genes associated with human solid tumors typicallyexert their effect by overexpression or altered expression. Geneamplification is a common mechanism leading to upregulation of geneexpression. Evidence from cytogenetic studies indicates that significantamplification occurs in over 50% of human breast cancers. Most notably,the amplification of the proto-oncogene human epidermal growth factorreceptor 2 (HER2) located on chromosome 17 (17(17q21-q22)), results inoverexpression of HER2 receptors on the cell surface leading toexcessive and dysregulated signaling in breast cancer and othermalignancies (Park et al., Clinical Breast Cancer 8:392-401[2008]). Avariety of oncogenes have been found to be amplified in other humanmalignancies. Examples of the amplification of cellular oncogenes inhuman tumors include amplifications of: c-myc in promyelocytic leukemiacell line HL60, and in small-cell lung carcinoma cell lines, N-myc inprimary neuroblastomas (stages III and IV), neuroblastoma cell lines,retinoblastoma cell line and primary tumors, and small-cell lungcarcinoma lines and tumors, L-myc in small-cell lung carcinoma celllines and tumors, c-myb in acute myeloid leukemia and in colon carcinomacell lines, c-erbb in epidermoid carcinoma cell, and primary gliomas,c-K-ras-2 in primary carcinomas of lung, colon, bladder, and rectum,N-ras in mammary carcinoma cell line (Varmus H., Ann Rev Genetics 18:553-612 (1984) [cited in Watson et al., Molecular Biology of the Gene(4th ed.; Benjamin/Cummings Publishing Co. 1987)].

Chromosomal deletions involving tumor suppressor genes may play animportant role in the development and progression of solid tumors. Theretinoblastoma tumor suppressor gene (Rb-1), located in chromosome13q14, is the most extensively characterized tumor suppressor gene. TheRb-1 gene product, a 105 kDa nuclear phosphoprotein, apparently plays animportant role in cell cycle regulation (Howe et al., Proc Natl Acad Sci(USA)87:5883-5887[1990]). Altered or lost expression of the Rb proteinis caused by inactivation of both gene alleles either through a pointmutation or a chromosomal deletion. Rb-i gene alterations have beenfound to be present not only in retinoblastomas but also in othermalignancies such as osteosarcomas, small cell lung cancer (Rygaard etal., Cancer Res 50: 5312-5317 [1990)]) and breast cancer. Restrictionfragment length polymorphism (RFLP) studies have indicated that suchtumor types have frequently lost heterozygosity at 13q suggesting thatone of the Rb-1 gene alleles has been lost due to a gross chromosomaldeletion (Bowcock et al., Am J Hum Genet, 46: 12 [1990]). Chromosome 1abnormalities including duplications, deletions and unbalancedtranslocations involving chromosome 6 and other partner chromosomesindicate that regions of chromosome 1, in particular 1q21-1q32 and1p11-13, might harbor oncogenes or tumor suppressor genes that arepathogenetically relevant to both chronic and advanced phases ofmyeloproliferative neoplasms (Caramazza et al., Eur J Hematol84:191-200[2010]). Myeloproliferative neoplasms are also associated withdeletions of chromosome 5. Complete loss or interstitial deletions ofchromosome 5 are the most common karyotypic abnormality inmyelodysplastic syndromes (MDSs). Isolated del(5q)/5q-MDS patients havea more favorable prognosis than those with additional karyotypicdefects, who tend to develop myeloproliferative neoplasms (MPNs) andacute myeloid leukemia. The frequency of unbalanced chromosome 5deletions has led to the idea that 5q harbors one or moretumor-suppressor genes that have fundamental roles in the growth controlof hematopoietic stem/progenitor cells (HSCs/HPCs). Cytogenetic mappingof commonly deleted regions (CDRs) centered on 5q31 and 5q32 identifiedcandidate tumor-suppressor genes, including the ribosomal subunit RPS14,the transcription factor Egr1/Krox20 and the cytoskeletal remodelingprotein, alpha-catenin (Eisenmann et al., Oncogene 28:3429-3441[2009]).Cytogenetic and allelotyping studies of fresh tumours and tumour celllines have shown that allelic loss from several distinct regions onchromosome 3p, including 3p25, 3p21-22, 3p21.3, 3p12-13 and 3p14, arethe earliest and most frequent genomic abnormalities involved in a widespectrum of major epithelial cancers of lung, breast, kidney, head andneck, ovary, cervix, colon, pancreas, esophagous, bladder and otherorgans. Several tumor suppressor genes have been mapped to thechromosome 3p region, and are thought that interstitial deletions orpromoter hypermethylation precede the loss of the 3p or the entirechromosome 3 in the development of carcinomas (Angeloni D., BriefingsFunctional Genomics 6:19-39[2007]).

Newborns and children with Down syndrome (DS) often present withcongenital transient leukemia and have an increased risk of acutemyeloid leukemia and acute lymphoblastic leukemia. Chromosome 21,harboring about 300 genes, may be involved in numerous structuralaberrations, e.g., translocations, deletions, and amplifications, inleukemias, lymphomas, and solid tumors. Moreover, genes located onchromosome 21 have been identified that play an important role intumorigenesis. Somatic numerical as well as structural chromosome 21aberrations are associated with leukemias, and specific genes includingRUNX1, TMPRSS2, and TFF, which are located in 21q, play a role intumorigenesis (Fonatsch C Gene Chromosomes Cancer 49:497-508[2010]).

In one embodiment, the method provides a means to assess the associationbetween gene amplification and the extent of tumor evolution.Correlation between amplification and/or deletion and stage or grade ofa cancer may be prognostically important because such information maycontribute to the definition of a genetically based tumor grade thatwould better predict the future course of disease with more advancedtumors having the worst prognosis. In addition, information about earlyamplification and/or deletion events may be useful in associating thoseevents as predictors of subsequent disease progression. Geneamplification and deletions as identified by the method can beassociated with other known parameters such as tumor grade, histology,Brd/Urd labeling index, hormonal status, nodal involvement, tumor size,survival duration and other tumor properties available fromepidemiological and biostatistical studies. For example, tumor DNA to betested by the method could include atypical hyperplasia, ductalcarcinoma in situ, stage I-III cancer and metastatic lymph nodes inorder to permit the identification of associations betweenamplifications and deletions and stage. The associations made may makepossible effective therapeutic intervention. For example, consistentlyamplified regions may contain an overexpressed gene, the product ofwhich may be able to be attacked therapeutically (for example, thegrowth factor receptor tyrosine kinase, p185^(HER2)).

The method can be used to identify amplification and/or deletion eventsthat are associated with drug resistance by determining the copy numbervariation of nucleic acids from primary cancers to those of cells thathave metastasized to other sites. If gene amplification and/or deletionis a manifestation of karyotypic instability that allows rapiddevelopment of drug resistance, more amplification and/or deletion inprimary tumors from chemoresistant patients than in tumors inchemosensitive patients would be expected. For example, if amplificationof specific genes is responsible for the development of drug resistance,regions surrounding those genes would be expected to be amplifiedconsistently in tumor cells from pleural effusions of chemoresistantpatients but not in the primary tumors. Discovery of associationsbetween gene amplification and/or deletion and the development of drugresistance may allow the identification of patients that will or willnot benefit from adjuvant therapy.

In other embodiments, the present method can be used to identifypolymorphisms associated with trinucleotide repeat disorders, which area set of genetic disorders caused by trinucleotide repeat expansion.Trinucleotide expansions are a subset of unstable microsatellite repeatsthat occur throughout all genomic sequences. If the repeat is present ina healthy gene, a dynamic mutation may increase the repeat count andresult in a defective gene. In one embodiment, the method can be used toidentify trinucleotide repeats associated with fragile X syndrome. Thelong arm of the X chromosome of patients suffering from fragile Xsyndrome can contain from 230 to 4000 CGG, as compared with 60 to 230repeats in carriers and 5 to 54 repeats in unaffected individuals. Thechromosomal instability resulting from this trinucleotide expansionpresents clinically as mental retardation, distinctive facial features,and macroorchidism in males. The second, related DNA-triplet repeatdisease, fragile X-E syndrome, was also identified on the X chromosome,but was found to be the result of an expanded CCG repeat. The presentmethod can identify trinucleotide repeats associated with other repeatexpansion disorders including Categories I, II and III. Category Idisorders include Huntington's disease (HD) and the spinocerebellarataxias that are caused by a CAG repeat expansion in protein-codingportions of specific genes. Category II expansions tend to be morephenotypically diverse with heterogeneous expansions that are generallysmall in magnitude, but also found in the exons of genes. Category IIIincludes fragile X syndrome, myotonic dystrophy, two of thespinocerebellar ataxias, juvenile myoclonic epilepsy and friereich'sataxia. These diseases are characterized by typically much larger repeatexpansions than the first two groups, and the repeats are locatedoutside of the protein-coding regions of the genes.

In other embodiments, the present method can identify CAG trinucleotiderepeats associated with at least ten neurologic disorders known to becaused by an increased number of CAG repeats, typically in codingregions of otherwise unrelated proteins. During protein synthesis, theexpanded CAG repeats are translated into a series of uninterruptedglutamine residues forming what is known as a polyglutamine tract(“polyQ”). Such polyglutamine tracts may be subject to increasedaggregation. These disorders are characterized by autosomal dominantmode of inheritance (with the exception of spino-bulbar muscular atrophywhich shows X-linked inheritance), midlife onset, a progressive course,and a correlation of the number of CAG repeats with the severity ofdisease and the age at onset. Causative genes are widely expressed inall of the known polyglutamine diseases. A common symptom of PolyQdiseases is characterized by a progressive degeneration of nerve cellsusually affecting people later in life. Although these diseases sharethe same repeated codon (CAG) and some symptoms, the repeats for thedifferent polyglutamine diseases occur on different chromosomes.Examples of polyQ disorders that can be identified by the present methodinclude without limitation DRPLA (Dentatorubropallidoluysian atrophy),HD (Huntington's disease), SBMA (Spinobulbar muscular atrophy or Kennedydisease), SCA1 (Spinocerebellar ataxia Type 1), SCA2 (Spinocerebellarataxia Type 2), SCA3 (Spinocerebellar ataxia Type 3 or Machado-Josephdisease), SCA6 (Spinocerebellar ataxia Type 6), SCAT (Spinocerebellarataxia Type 7), SCA17 (Spinocerebellar ataxia Type 17). Examples ofnon-polyQ disorders that can be identified by the present method includeFRAXA (Fragile X syndrome), FXTAS (Fragile X-associated tremor/ataxiasyndrome), FRAXE (Fragile XE mental retardation), FRDA (Friedreich'sataxia), DM (Myotonic dystrophy), SCA8 (Spinocerebellar ataxia Type 8),SCA12 (Spinocerebellar ataxia Type 12).

In addition to the role of CNV in cancer, CNVs have been associated witha growing number of common complex disease, including humanimmunodeficiency virus (HIV), autoimmune diseases and a spectrum ofneuropsychiatric disorders.

To date a number of studies have reported association between CNV ingenes involved in inflammation and the immune response and HIV, asthma,Crohn's disease and other autoimmune disorders (Fanciulli et al., ClinGenet 77:201-213[2010]). For example, CNV in CCL3L1, has been implicatedin HIV/AIDS susceptibility (CCL3L1, 17q11.2 deletion), rheumatoidarthritis (CCL3L1, 17q11.2 deletion), and Kawasaki disease (CCL3L1,17q11.2 duplication); CNV in HBD-2, has been reported to predispose tocolonic Crohn's disease (HDB-2, 8p23.1 deletion) and psoriasis (HDB-2,8p23.1 deletion); CNV in FCGR3B, was shown to predispose toglomerulonephritis in systemic lupus erthematosous (FCGR3B, 1q23deletion, 1q23 duplication), anti-neutrophil cytoplasmic antibody(ANCA)-associated vasculatis (FCGR3B, 1q23 deletion), and increase therisk of developing rheumatoid arthritis. There are at least twoinflammatory or autoimmune diseases that have been shown to beassociated with CNV at different gene loci. For example, Crohn's diseaseis associated with low copy number at HDB-2, but also with a commondeletion polymorphism upstream of the IGRM gene that encodes a member ofthe p47 immunity-related GTPase family. In addition to the associationwith FCGR3B copy number, SLE susceptibility has also been reported to besignificantly increased among subjects with a lower number of copies ofcomplement component C4.

Associations between genomic deletions at the GSTM1 (GSTM1,1q23deletion) and GSTT1 (GSTT1, 22q11.2 deletion) loci and increasedrisk of atopic asthma have been reported in a number of independentstudies. In some embodiments, the present method can be used todetermine the presence or absence of a CNV associated with inflammationand/or autoimmune diseases. For example, the present method can be usedto determine the presence of a CNV in a patient suspected to besuffering from HIV, asthma, or Crohn's disease. Examples of CNVassociated with such diseases include without limitation deletions at17q11.2, 8p23.1, 1q23, and 22q11.2, and duplications at 17q11.2, and1q23. In some embodiments, the present method can be used to determinethe presence of CNV in genes including but not limited to CCL3L1, HBD-2,FCGR3B, GSTM, GSTT1, C4, and MGM.

Associations between de novo and inherited CNV and several commonneurological and psychiatric diseases have been reported in autism,schizophrenia and epilepsy, and some cases of neurodegenerative diseasessuch as Parkinson's disease, amyotrophic lateral sclerosis (ALS) andautosomal dominant Alzheimer's disease (Fanciulli et al., Clin Genet77:201-213[2010]). Cytogenetic abnormalities have been observed inpatients with autism and autism spectrum disorders (ASDs) withduplications at 15q11-q13. According to the Autism Genome projectConsortium, 154 CNV including several recurrent CNVs, either onchromosome 15q11-q13 or at new genomic locations including chromosome2p16, 1q21 and at 17p12 in a region associated with Smith-Magenissyndrome that overlaps with ASD. Recurrent microdeletions ormicroduplications on chromosome 16p11.2 have highlighted the observationthat de novo CNVs are detected at loci for genes such as SHANK3 (22q13.3deletion), neurexin 1 (NRXNJ, 2p16.3 deletion) and the neuroglins(NLGN4, Xp22.33 deletion) that are known to regulate synapticdifferentiation and regulate glutaminergic neurotransmitter release.Schizophrenia has also been associated with multiple de novo CNVs.Microdeletions and microduplications associated with schizophreniacontain an overrepresentation of genes belonging to neurodevelopmentaland glutaminergic pathways, suggesting that multiple CNVs affectingthese genes may contribute directly to the pathogenesis of schizophreniae.g. ERBB4, 2q34 deletion, SLC1A3, 5p13.3 deletion; RAPEGF4, 2q31.1deletion; CIT, 12.24 deletion; and multiple genes with de novo CNV. CNVshave also been associated with other neurological disorders includingepilepsy (CHRNA7, 15q13.3 deletion), Parkinson's disease (SNCA 4q22duplication) and ALS (SMN1, 5q12.2.-q13.3 deletion; and SMN2 deletion).In some embodiments, the present method can be used to determine thepresence or absence of a CNV associated with diseases of the nervoussystem. For example, the present method can be used to determine thepresence of a CNV in a patient suspected to be suffering from autism,schizophrenia, epilepsy, neurodegenerative diseases such as Parkinson'sdisease, amyotrophic lateral sclerosis (ALS) or autosomal dominantAlzheimer's disease. The present method can be used to determine CNV ofgenes associated with diseases of the nervous system including withoutlimitation any of the Autism Spectrum Disorders (ASD), schizophrenia,and epilepsy, and CNV of genes associated with neurodegenerativedisorders such as Parkinson's disease. Examples of CNV associated withsuch diseases include without limitation duplications at 15q11-q13,2p16, 1q21, 17p12, 16p11.2, and 4q22, and deletions at 22q13.3, 2p16.3,Xp22.33, 2q34, 5p13.3, 2q31.1, 12.24, 15q13.3, and 5q12.2. In someembodiments, the present method can be used to determine the presence ofCNV in genes including but not limited to SHANK3, NLGN4, NRXN1, ERBB4,SLC1A3, RAPGEF4, CIT, CHRNA7, SNCA, SMN1, and SMN2.

The association between metabolic and cardiovascular traits, such asfamilial hypercholesterolemia (FH), atherosclerosis and coronary arterydisease, and CNVs has been reported in a number of studies (Fanciulli etal., Clin Genet 77:201-213 [2010]). For example, germlinerearrangements, mainly deletions, have been observed at the LDLR gene(LDLR, 19p13.2 deletion/duplication) in some FH patients who carry noother LDLR mutations. Another example is the LPA gene that encodesapolipoprotein(a) (apo(a)) whose plasma concentration is associated withrisk of coronary artery disease, myocardial infarction (MI) and stroke.Plasma concentrations of the apo(a) containing lipoprotein Lp(a) varyover 1000-fold between individuals and 90% of this variability isgenetically determined at the LPA locus, with plasma concentration andLp(a) isoform size being proportional to a highly variable number of‘kringle 4’ repeat sequences (range 5-50). These data indicate that CNVin at least two genes can be associated with cardiovascular risk. Thepresent method can be used in large studies to search specifically forCNV associations with cardiovascular disorders. In some embodiments, thepresent method can be used to determine the presence or absence of a CNVassociated with metabolic or cardiovascular disease. For example, thepresent method can be used to determine the presence of a CNV in apatient suspected to be suffering from familial hypercholesterolemia.The present method can be used to determine CNV of genes associated withmetabolic or cardiovascular disease e.g. hypercholesterolemia. Examplesof CNV associated with such diseases include without limitation 19p13.2deletion/duplication of the LDLR gene, and multiplications in the LPAgene.

Sequencing

In various embodiments, the method described herein employs nextgeneration sequencing technology (NGS) in which clonally amplified DNAtemplates or single DNA molecules are sequenced in a massively parallelfashion within a flow cell (e.g. as described in Volkerding et al. ClinChem 55:641-658[2009]; Metzker M Nature Rev 11:31-46[2010]). In additionto high-throughput sequence information, NGS provides digitalquantitative information, in that each sequence read is a countable“sequence tag” representing an individual clonal DNA template or asingle DNA molecule. The sequencing technologies of NGS includepyrosequencing, sequencing-by-synthesis with reversible dye terminators,sequencing by oligonucleotide probe ligation and real time sequencing.

In various embodiments, one can analyze samples that are not amplified,or are only partially amplified (target amplification). In some cases,the methods of determining fetal fraction can be accomplished withoutrequiring any type of targeted amplification.

Whole genome amplification that occurs as part of the sequencing processprovides sufficient copies that can be covered by increasing number ofsequencing cycles to provide increasingly better coverage.

In preferred embodiments, the sample comprising the mixture of DNAmolecules derived from two different genomes is non-specificallyenriched for the whole genome sequences prior to whole genome sequencingi.e. whole genome amplification is performed prior to sequencing.

Non-specific enrichment of sample DNA may refer to the whole genomeamplification of the genomic DNA fragments of the sample that can beused to increase the level of the sample DNA prior to identifyingpolymorphisms by sequencing. Non-specific enrichment can be theselective enrichment of one of the two genomes present in the sample.For example, non-specific enrichment can be selective of the fetalgenome in a maternal sample, which can be obtained by known methods toincrease the relative proportion of fetal to maternal DNA in a sample.Alternatively, non-specific enrichment can be the non-selectiveamplification of both genomes present in the sample. For example,non-specific amplification can be of fetal and maternal DNA in a samplecomprising a mixture of DNA from the fetal and maternal genomes. Methodsfor whole genome amplification are known in the art. Degenerateoligonucleotide-primed PCR (DOP), primer extension PCR technique (PEP)and multiple displacement amplification (MDA), are examples of wholegenome amplification methods. In some embodiments, the sample comprisingthe mixture of cfDNA from different genomes is unenriched for cfDNA ofthe genomes present in the mixture. In other embodiments, the samplecomprising the mixture of cfDNA from different genomes isnon-specifically enriched for any one of the genomes present in thesample.

In other embodiments, the cfDNA in the sample is enriched specifically.Specific enrichment refers to the enrichment of a genomic sample forspecific sequences e.g. polymorphic target sequence, which are selectedfor amplification prior to sequencing the DNA sample. However, anadvantage of the disclosed embodiments is that targeted amplification isnot needed. Polymorphic

Some of the sequencing technologies are available commercially, such asthe sequencing-by-hybridization platform from Affymetrix Inc.(Sunnyvale, Calif.) and the sequencing-by-synthesis platforms from 454Life Sciences (Bradford, Conn.), Illumina/Solexa (Hayward, Calif.) andHelicos Biosciences (Cambridge, Mass.), and the sequencing-by-ligationplatform from Applied Biosystems (Foster City, Calif.), as describedbelow. In addition to the single molecule sequencing performed usingsequencing-by-synthesis of Helicos Biosciences, other single moleculesequencing technologies are encompassed by the disclosed method andinclude the SMRT™ technology of Pacific Biosciences, the Ion Torrent™technology, and nanopore sequencing being developed for example, byOxford Nanopore Technologies.

While the automated Sanger method is considered as a ‘first generation’technology, Sanger sequencing including the automated Sanger sequencing,can also be employed by the disclosed method. Additional sequencingmethods that comprise the use of developing nucleic acid imagingtechnologies e.g. atomic force microscopy (AFM) or transmission electronmicroscopy (TEM), are also encompassed by the disclosed method.Exemplary sequencing technologies are described below.

In one embodiment, the DNA sequencing technology that is used in thedisclosed methods is the Helicos True Single Molecule Sequencing (tSMS)(e.g. as described in Harris T. D. et al., Science 320:106-109 [2008]).In the tSMS technique, a DNA sample is cleaved into strands ofapproximately 100 to 200 nucleotides, and a polyA sequence is added tothe 3′ end of each DNA strand. Each strand is labeled by the addition ofa fluorescently labeled adenosine nucleotide. The DNA strands are thenhybridized to a flow cell, which contains millions of oligo-T capturesites that are immobilized to the flow cell surface. The templates canbe at a density of about 100 million templates/cm². The flow cell isthen loaded into an instrument, e.g., HeliScope™ sequencer, and a laserilluminates the surface of the flow cell, revealing the position of eachtemplate. A CCD camera can map the position of the templates on the flowcell surface. The template fluorescent label is then cleaved and washedaway. The sequencing reaction begins by introducing a DNA polymerase anda fluorescently labeled nucleotide. The oligo-T nucleic acid serves as aprimer. The polymerase incorporates the labeled nucleotides to theprimer in a template directed manner. The polymerase and unincorporatednucleotides are removed. The templates that have directed incorporationof the fluorescently labeled nucleotide are discerned by imaging theflow cell surface. After imaging, a cleavage step removes thefluorescent label, and the process is repeated with other fluorescentlylabeled nucleotides until the desired read length is achieved. Sequenceinformation is collected with each nucleotide addition step. Wholegenome sequencing by single molecule sequencing technologies excludesPCR-based amplification in the preparation of the sequencing libraries,and the directness of sample preparation allows for direct measurementof the sample, rather than measurement of copies of that sample.

In one embodiment, the DNA sequencing technology that is used in thedisclosed methods is the 454 sequencing (Roche) (e.g. as described inMargulies, M. et al. Nature 437:376-380 (2005)). 454 sequencing involvestwo steps. In the first step, DNA is sheared into fragments ofapproximately 300-800 base pairs, and the fragments are blunt-ended.Oligonucleotide adaptors are then ligated to the ends of the fragments.The adaptors serve as primers for amplification and sequencing of thefragments. The fragments can be attached to DNA capture beads, e.g.,streptavidin-coated beads using, e.g., Adaptor B, which contains5′-biotin tag. The fragments attached to the beads are PCR amplifiedwithin droplets of an oil-water emulsion. The result is multiple copiesof clonally amplified DNA fragments on each bead. In the second step,the beads are captured in wells (pico-liter sized). Pyrosequencing isperformed on each DNA fragment in parallel. Addition of one or morenucleotides generates a light signal that is recorded by a CCD camera ina sequencing instrument. The signal strength is proportional to thenumber of nucleotides incorporated. Pyrosequencing makes use ofpyrophosphate (PPi) which is released upon nucleotide addition. PPi isconverted to ATP by ATP sulfurylase in the presence of adenosine 5′phosphosulfate. Luciferase uses ATP to convert luciferin tooxyluciferin, and this reaction generates light that is discerned andanalyzed.

In one embodiment, the DNA sequencing technology that is used in thedisclosed methods is the SOLiD™ technology (Applied Biosystems). InSOLiD™ sequencing-by-ligation, genomic DNA is sheared into fragments,and adaptors are attached to the 5′ and 3′ ends of the fragments togenerate a fragment library. Alternatively, internal adaptors can beintroduced by ligating adaptors to the 5′ and 3′ ends of the fragments,circularizing the fragments, digesting the circularized fragment togenerate an internal adaptor, and attaching adaptors to the 5′ and 3′ends of the resulting fragments to generate a mate-paired library. Next,clonal bead populations are prepared in microreactors containing beads,primers, template, and PCR components. Following PCR, the templates aredenatured and beads are enriched to separate the beads with extendedtemplates. Templates on the selected beads are subjected to a 3′modification that permits bonding to a glass slide. The sequence can bedetermined by sequential hybridization and ligation of partially randomoligonucleotides with a central determined base (or pair of bases) thatis identified by a specific fluorophore. After a color is recorded, theligated oligonucleotide is cleaved and removed and the process is thenrepeated.

In one embodiment, the DNA sequencing technology that is used in thedisclosed methods is the single molecule, real-time (SMRT™) sequencingtechnology of Pacific Biosciences. In SMRT sequencing, the continuousincorporation of dye-labeled nucleotides is imaged during DNA synthesis.Single DNA polymerase molecules are attached to the bottom surface ofindividual zero-mode wavelength identifiers (ZMW identifiers) thatobtain sequence information while phospholinked nucleotides are beingincorporated into the growing primer strand. A ZMW is a confinementstructure which enables observation of incorporation of a singlenucleotide by DNA polymerase against the background of fluorescentnucleotides that rapidly diffuse in an out of the ZMW (in microseconds).It takes several milliseconds to incorporate a nucleotide into a growingstrand. During this time, the fluorescent label is excited and producesa fluorescent signal, and the fluorescent tag is cleaved off.Identification of the corresponding fluorescence of the dye indicateswhich base was incorporated. The process is repeated.

In one embodiment, the DNA sequencing technology that is used in thedisclosed methods is nanopore sequencing (e.g. as described in Soni G Vand Meller A. Clin Chem 53: 1996-2001 [2007]). Nanopore sequencing DNAanalysis techniques are being industrially developed by a number ofcompanies, including Oxford Nanopore Technologies (Oxford, UnitedKingdom). Nanopore sequencing is a single-molecule sequencing technologywhereby a single molecule of DNA is sequenced directly as it passesthrough a nanopore. A nanopore is a small hole, of the order of 1nanometer in diameter. Immersion of a nanopore in a conducting fluid andapplication of a potential (voltage) across it results in a slightelectrical current due to conduction of ions through the nanopore. Theamount of current which flows is sensitive to the size and shape of thenanopore. As a DNA molecule passes through a nanopore, each nucleotideon the DNA molecule obstructs the nanopore to a different degree,changing the magnitude of the current through the nanopore in differentdegrees. Thus, this change in the current as the DNA molecule passesthrough the nanopore represents a reading of the DNA sequence.

In one embodiment, the DNA sequencing technology that is used in thedisclosed methods is the chemical-sensitive field effect transistor(chemFET) array (e.g., as described in U.S. Patent Publication No.2009/0026082 filed Dec. 17, 2007). In one example of the technique, DNAmolecules can be placed into reaction chambers, and the templatemolecules can be hybridized to a sequencing primer bound to apolymerase. Incorporation of one or more triphosphates into a newnucleic acid strand at the 3′ end of the sequencing primer can bediscerned by a change in current by a chemFET. An array can havemultiple chemFET sensors. In another example, single nucleic acids canbe attached to beads, and the nucleic acids can be amplified on thebead, and the individual beads can be transferred to individual reactionchambers on a chemFET array, with each chamber having a chemFET sensor,and the nucleic acids can be sequenced.

In one embodiment, the DNA sequencing technology that is used in thedisclosed methods is the Halcyon Molecular's method that usestransmission electron microscopy (TEM). The method, termed IndividualMolecule Placement Rapid Nano Transfer (IMPRNT), comprises utilizingsingle atom resolution transmission electron microscope imaging ofhigh-molecular weight (150 kb or greater) DNA selectively labeled withheavy atom markers and arranging these molecules on ultra-thin films inultra-dense (3 nm strand-to-strand) parallel arrays with consistentbase-to-base spacing. The electron microscope is used to image themolecules on the films to determine the position of the heavy atommarkers and to extract base sequence information from the DNA. Themethod is further described in PCT patent publication WO 2009/046445.The method allows for sequencing complete human genomes in less than tenminutes.

In one embodiment, the DNA sequencing technology is the Ion Torrentsingle molecule sequencing, which pairs semiconductor technology with asimple sequencing chemistry to directly translate chemically encodedinformation (A, C, G, T) into digital information (0, 1) on asemiconductor chip. In nature, when a nucleotide is incorporated into astrand of DNA by a polymerase, a hydrogen ion is released as abyproduct. Ion Torrent uses a high-density array of micro-machined wellsto perform this biochemical process in a massively parallel way. Eachwell holds a different DNA molecule. Beneath the wells is anion-sensitive layer and beneath that an ion sensor. When a nucleotide,for example a C, is added to a DNA template and is then incorporatedinto a strand of DNA, a hydrogen ion will be released. The charge fromthat ion will change the pH of the solution, which can be identified byIon Torrent's ion sensor. The sequencer—essentially the world's smallestsolid-state pH meter—calls the base, going directly from chemicalinformation to digital information. The Ion personal Genome Machine(PGM™) sequencer then sequentially floods the chip with one nucleotideafter another. If the next nucleotide that floods the chip is not amatch. No voltage change will be recorded and no base will be called. Ifthere are two identical bases on the DNA strand, the voltage will bedouble, and the chip will record two identical bases called. Directidentification allows recordation of nucleotide incorporation inseconds.

In some embodiments, the methods employ PCR or a related technique foramplifying sample nucleotide sequences before identifying or mappingthem. However, the algorithmic techniques disclosed herein generally donot require amplification, particularly targeted amplification ofpolymorphisms used to estimate genome fraction.

Certain embodiments employ digital PCR and sequencing by hybridization.Digital polymerase chain reaction (digital PCR or dPCR) can be used todirectly identify and quantify nucleic acids in a sample. Digital PCRcan be performed in an emulsion. Individual nucleic acids are separated,e.g., in a microfluidic chamber device, and each nucleic acid isindividually amplified by PCR. Nucleic acids can be separated such thereis an average of approximately 0.5 nucleic acids/well, or not more thanone nucleic acid/well. Different probes can be used to distinguish fetalalleles and maternal alleles. Alleles can be enumerated to determinecopy number. In sequencing by hybridization, the hybridization comprisescontacting the plurality of polynucleotide sequences with a plurality ofpolynucleotide probes, wherein each of the plurality of polynucleotideprobes can be optionally tethered to a substrate. The substrate might beflat surface comprising an array of known nucleotide sequences. Thepattern of hybridization to the array can be used to determine thepolynucleotide sequences present in the sample. In other embodiments,each probe is tethered to a bead, e.g., a magnetic bead or the like.Hybridization to the beads can be identified and used to identify theplurality of polynucleotide sequences within the sample.

In one embodiment, the method employs massively parallel sequencing ofmillions of DNA fragments using Illumina's sequencing-by-synthesis andreversible terminator-based sequencing chemistry (e.g. as described inBentley et al., Nature 6:53-59[2009]). Template DNA can be genomic DNAe.g. cfDNA. In some embodiments, genomic DNA from isolated cells is usedas the template, and it is fragmented into lengths of several hundredbase pairs. In other embodiments, cfDNA is used as the template, andfragmentation is not required as cfDNA exists as short fragments. Forexample fetal cfDNA circulates in the bloodstream as fragments of <300bp, and maternal cfDNA has been estimated to circulate as fragments ofbetween about 0.5 and 1 Kb (Li et al., Clin Chem, 50: 1002-1011 (2004)).Illumina's sequencing technology relies on the attachment of fragmentedgenomic DNA to a planar, optically transparent surface on whicholigonucleotide anchors are bound. Template DNA is end-repaired togenerate 5′-phosphorylated blunt ends, and the polymerase activity ofKlenow fragment is used to add a singleA base to the 3′ end of the bluntphosphorylated DNA fragments. This addition prepares the DNA fragmentsfor ligation to oligonucleotide adapters, which have an overhang of asingle T base at their 3′ end to increase ligation efficiency. Theadapter oligonucleotides are complementary to the flow-cell anchors.Under limiting-dilution conditions, adapter-modified, single-strandedtemplate DNA is added to the flow cell and immobilized by hybridizationto the anchors. Attached DNA fragments are extended and bridge amplifiedto create an ultra-high density sequencing flow cell with hundreds ofmillions of clusters, each containing ˜1,000 copies of the sametemplate. In one embodiment, the randomly fragmented genomic DNA e.g.cfDNA, is amplified using PCR before it is subjected to clusteramplification. Alternatively, an amplification-free genomic librarypreparation is used, and the randomly fragmented genomic DNA e.g. cfDNAis enriched using the cluster amplification alone (Kozarewa et al.,Nature Methods 6:291-295[2009]). The templates are sequenced using arobust four-color DNA sequencing-by-synthesis technology that employsreversible terminators with removable fluorescent dyes. High-sensitivityfluorescence identification is achieved using laser excitation and totalinternal reflection optics. Short sequence reads of about 20-40 bp e.g.36 bp, are aligned against a repeat-masked reference genome and geneticdifferences are called using specially developed data analysis pipelinesoftware. After completion of the first read, the templates can beregenerated in situ to enable a second read from the opposite end of thefragments. Thus, either single-end or paired end sequencing of the DNAfragments is used according to the method. Partial sequencing of DNAfragments present in the sample is performed, and sequence tagscomprising reads of predetermined length e.g. 36 bp, that are mapped toa known reference genome are counted.

The length of the sequence read is associated with the particularsequencing technology. NGS methods provide sequence reads that vary insize from tens to hundreds of base pairs. In some embodiments of themethod described herein, the sequence reads are about 20 bp, about 25bp, about 30 bp, about 35 bp, about 40 bp, about 45 bp, about 50 bp,about 55 bp, about 60 bp, about 65 bp, about 70 bp, about 75 bp, about80 bp, about 85 bp, about 90 bp, about 95 bp, about 100 bp, about 110bp, about 120 bp, about 130, about 140 bp, about 150 bp, about 200 bp,about 250 bp, about 300 bp, about 350 bp, about 400 bp, about 450 bp, orabout 500 bp. It is expected that technological advances will enablesingle-end reads of greater than 500 bp enabling for reads of greaterthan about 1000 bp when paired end reads are generated. In oneembodiment, the sequence reads are 36 bp. Other sequencing methods thatcan be employed by the disclosed methods include the single moleculesequencing methods that can sequence nucleic acids molecules>5000 bp.The massive quantity of sequence output is transferred by an analysispipeline that transforms primary imaging output from the sequencer intostrings of bases. A package of integrated algorithms performs the coreprimary data transformation steps: image analysis, intensity scoring,base calling, and alignment.

Mapping

Various computational methods can be used to map each identifiedsequence to a bin, e.g., by identifying all sequences in the sample thatmap to a particular gene, chromosome, allele, or other structure. Anumber of computer algorithms exist to align sequences, includingwithout limitation BLAST (Altschul et al., 1990), BLITZ (MPsrch)(Sturrock& Collins, 1993), FASTA (Person &Lipman, 1988), BOWTIE(Langmead et al., Genome Biology 10:R25.1-R25.10[2009]), or ELAND(Illumina, Inc., San Diego, Calif., USA), In some embodiments, thesequences of the bins are found in nucleic acid databases known to thosein the art, including without limitation GenBank, dbEST, dbSTS, EMBL(the European Molecular Biology Laboratory), and the DDBJ (the DNADatabank of Japan). BLAST or similar tools can be used to search theidentified sequences against the sequence databases, and search hits canbe used to sort the identified sequences into the appropriate bins.

Apparatus

Analysis of the sequencing data and the diagnoses derived therefrom aretypically performed using computer hardware operating according todefined algorithms and programs. Therefore, certain embodiments employprocesses involving data stored in or transferred through one or morecomputer systems or other processing systems. Embodiments of theinvention also relate to apparatus for performing these operations. Thisapparatus may be specially constructed for the required purposes, or itmay be a general-purpose computer (or a group of computers) selectivelyactivated or reconfigured by a computer program and/or data structurestored in the computer. In some embodiments, a group of processorsperforms some or all of the recited analytical operationscollaboratively (e.g., via a network or cloud computing) and/or inparallel. A processor or group of processors for performing the methodsdescribed herein may be of various types including microcontrollers andmicroprocessors such as programmable devices (e.g., CPLDs and FPGAs) andother devices such as gate array ASICs, digital signal processors,and/or general purpose microprocessors.

In addition, certain embodiments relate to tangible and/ornon-transitory computer readable media or computer program products thatinclude program instructions and/or data (including data structures) forperforming various computer-implemented operations. Examples ofcomputer-readable media include, but are not limited to, semiconductormemory devices, magnetic media such as disk drives, magnetic tape,optical media such as CDs, magneto-optical media, and hardware devicesthat are specially configured to store and perform program instructions,such as read-only memory devices (ROM) and random access memory (RAM).The computer readable media may be directly controlled by an end user orthe media may be indirectly controlled by the end user. Examples ofdirectly controlled media include the media located at a user facilityand/or media that are not shared with other entities. Examples ofindirectly controlled media include media that is indirectly accessibleto the user via an external network and/or via a service providingshared resources such as the “cloud.” Examples of program instructionsinclude both machine code, such as produced by a compiler, and filescontaining higher level code that may be executed by the computer usingan interpreter.

In one embodiment, a computer program product is provided for generatingan output indicating the fraction of nucleic acid derived from a definedgenome (such as that of a fetus) and optionally other information suchas the presence or absence of a fetal aneuploidy in a test sample. Thecomputer product may contain instructions for performing any one or moreof the above-described methods for determining a fraction of nucleicacids from a particular organism. As explained, the computer product mayinclude a non-transitory and/or tangible computer readable medium havinga computer executable or compilable logic (e.g., instructions) recordedthereon for enabling a processor to determine genome fraction and, insome cases, whether an aneuploidy or other condition is present orabsent in the genome. In one example, the computer product comprises acomputer readable medium having a computer executable or compilablelogic (e.g., instructions) recorded thereon for enabling a processor todetermine fetal fraction and diagnose a fetal aneuploidy comprising: areceiving procedure for receiving sequencing data from at least aportion of nucleic acid molecules from a maternal biological sample,wherein said sequencing data comprises sequences at the loci of one ormore polymorphisms; computer assisted logic for analyzing sequences todetermine allele counts for the one or more polymorphisms, anddetermining fetal fraction of the nucleic acids in the maternalbiological sample; and an output procedure for generating an outputindicating the fetal fraction of nucleic acids in the sample.

The sequence information from the sample under consideration may bemapped to polymorphism reference sequences as described. Further, themapped sequence information may be used to generate allele counts and/ordetermine zygosity cases for the polymorphisms. Such information may beused to determine fetal fraction. In various embodiments, polymorphismreference sequences are stored in a database such as a relational orobject database, for example. It should be understood that it is notpractical, or even possible in most cases, for an unaided human being toperform any one or all of these computational operations. For example,mapping a single 30 bp read from a sample to a database of polymorphismreference sequences will take potentially a prohibitively long periodwithout the assistance of a computational apparatus. Of course, theproblem is compounded because reliable calls often require mappingthousands (e.g., at least about 10,000) or even millions of reads to oneor more chromosomes.

In certain embodiments, the disclosed methods make use of a stored listor other organized collection of data concerning reference polymorphismsfor the organism producing the nucleic acid sequences to be analyzed. Asexplained above, the sequences from the sample under consideration canbe aligned or otherwise mapped to the stored polymorphisms. Theindividual polymorphisms are typically sequences of a length sufficientto unambiguously map to sequences identified from the nucleic acidsample. Typically, the polymorphisms come in groups, one for eachallele. In various embodiments, the reference polymorphisms are storedin a database containing characteristics of the polymorphisms inaddition to their sequences. This collection of information aboutpolymorphisms may be stored in a relational or object database, forexample.

FIG. 10 illustrates a typical computer system that, when appropriatelyconfigured or designed, can serve as an analysis apparatus of thisinvention. The computer system 200 includes any number of processors 202(also referred to as central processing units, or CPUs) that are coupledto storage devices including primary storage 206 (typically a randomaccess memory, or RAM), primary storage 204 (typically a read onlymemory, or ROM). CPU 202 may be of various types includingmicrocontrollers and microprocessors such as programmable devices (e.g.,CPLDs and FPGAs) and unprogrammable devices such as gate array ASICs orgeneral purpose microprocessors. As is well known in the art, primarystorage 204 acts to transfer data and instructions to the CPU andprimary storage 206 is used typically to transfer data and instructionsin a bi-directional manner. Both of these primary storage devices mayinclude any suitable computer-readable media such as those describedabove. A mass storage device 208 is also coupled bi-directionally to CPU202 and provides additional data storage capacity and may include any ofthe computer-readable media described above. Mass storage device 208 maybe used to store programs, data and the like and is typically asecondary storage medium such as a hard disk. It will be appreciatedthat the information retained within the mass storage device 208, may,in appropriate cases, be incorporated in standard fashion as part ofprimary storage 206 as virtual memory. A specific mass storage devicesuch as a CD-ROM 214 may also pass data uni-directionally to the CPU.

CPU 202 is also coupled to an interface 210 that connects to one or moreinput/output devices such as such as video monitors, mice, keyboards,microphones, touch-sensitive displays, transducer card readers, tablets,styluses, voice or handwriting recognizers, or other well-known inputdevices such as, of course, other computers. Finally, CPU 202 optionallymay be coupled to an external device such as a database or a computer ortelecommunications network using an external connection as showngenerally at 212. With such a connection, it is contemplated that theCPU might receive information from the network, or might outputinformation to the network in the course of performing the method stepsdescribed herein.

Sequence or other data, can be input into a computer by a user eitherdirectly or indirectly. In one embodiment, the computer system 200 isdirectly coupled to a sequencing tool that reads and/or analyzessequences of amplified nucleic acids. Sequences or other informationfrom such tools are provided via interface 212 for analysis by system200. Alternatively, the sequences processed by system 200 are providedfrom a sequence storage source such as a database or other repository.Once in the processing apparatus 200, a memory device such as primarystorage 206 or mass storage 208 buffers or stores, at least temporarily,sequences of the nucleic acids. In addition, the memory device may storetag numbers for various chromosomes or genes, calculated copy counts,etc. The memory may also store various routines and/or programs foranalyzing the presenting the sequence or mapped data. Suchprograms/routines may include programs for performing statisticalanalyses, etc.

In one example, a user provides a sample into a sequencing apparatus.Data is collected and/or analyzed by the sequencing apparatus which isconnected to a computer. Software on the computer allows for datacollection and/or analysis. Data can be stored, displayed (via a monitoror other similar device), and/or sent to another location. As indicated,the computer may be connected to the internet which is used to transmitdata to a handheld device utilized by a remote user (e.g., a physician,scientist or analyst). It is understood that the data can be storedand/or analyzed prior to transmittal. In some embodiments, raw data iscollected and sent to a remote user (or apparatus) who will analyzeand/or store the data. Transmittal can occur via the internet, but canalso occur via satellite or other connection. Alternately, data can bestored on a computer-readable medium (e.g., CD or semiconductor memorystorage device) and the medium can be shipped to an end user (e.g., viamail). The remote user can be in the same or a different geographicallocation including, but not limited to a building, city, state, countryor continent.

In some embodiments, the methods of the invention further comprisecollecting data regarding a plurality of polynucleotide sequences andsending the data to a computer. For example, the computer can beconnected to laboratory equipment, e.g., a sample collection apparatus,a nucleotide amplification apparatus, a nucleotide sequencing apparatus,or a hybridization apparatus. The computer can then collect applicabledata gathered by the laboratory device. The data can be stored on acomputer at any step, e.g., while collected in real time, prior to thesending, during or in conjunction with the sending, or following thesending. The data can be stored on a computer-readable medium that canbe extracted from the computer. The data collected or stored can betransmitted from the computer to a remote location, e.g., via a localnetwork or a wide area network such as the internet.

In one aspect, the invention further provides a system capable ofperforming quantitative analysis of nucleotide sequencing with aprecision of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or at least 99%. The nucleotide sequencing cancomprise Sanger sequencing, massively parallel sequencing, hybridizationor other techniques as described herein. The system can comprise variouscomponents, e.g., laboratory equipment and computer systems, and can beconfigured to carry out the methods of the invention disclosed herein.

In some embodiments, the apparatus and/or programming instructions mayfurther include instructions for automatically recording informationpertinent to the method such as fetal DNA fraction and optionally thepresence or absence of a fetal chromosomal aneuploidy in a patientmedical record for a human subject providing the maternal test sample.The patient medical record may be maintained by, for example, alaboratory, physician's office, a hospital, a health maintenanceorganization, an insurance company, or a personal medical recordwebsite. Further, based on the results of the processor-implementedanalysis, the method may further involve prescribing, initiating, and/oraltering treatment of a human subject from whom the maternal test samplewas taken. This may involve performing one or more additional tests oranalyses on additional samples taken from the subject.

EXAMPLE

Fetal Fraction Predicted from Sequenced Variations: Case 2

To demonstrate that the present method can be used to reliably estimatefetal fraction in a maternal sample, an artificial ‘maternal’ sample wascreated, and base variations were identified at all loci of chromosomes1 and 7 to predict the fraction of the minor contributing genome.

cfDNA that is isolated from a pregnant female is a mixture of maternaland fetal cfDNA, with the level of fetal cfDNA corresponding to a medianof ˜10% of the total cfDNA (Lo et al., 2010, “Maternal Plasma DNAsequencing reveals the genome-wide genetic and mutational profile of thefetus”, Prenatal Diagnosis, 2, 1-12). To create the artificial maternalsample, genomic DNA (gDNA) obtained from a mother and her son (motherand son DNAs NA10924 and NA10925; The Coriell Institute for MedicalResearch, Camden, N.J.) was used to create the sample of mixed genomes.Five micrograms of each of the mother's and son's gDNA were sheared intofragments of about 200 bp, and the concentration of each was determined.An artificial sample containing 10% DNA from the son and 90% DNA fromthe mother was created to imitate a maternal blood sample, which isbelieved to typically contain 2-40% fetal cfDNA, depending on thegestational age [Lun et al, 2008, “Microfluidics digital PCR reveals ahigher than expected fraction of fetal DNA in maternal plasma”, ClinicalChemistry, 54, 1664-1672]. A sequencing library was prepared from theDNA of the artificial sample, and subjected to 50 sequencing cycles on 4lanes of the flow cell using the IlluminaHiSeq 2000. Approximately 800million 49-mer sequence reads were generated.

The ˜800 million reads were aligned to the Repeat-masked Human ReferenceGenome (hg19 build) using the GSNAP algorithm(http://research-pub.gene.com/gmap/), allowing for one mismatch and noinsertions and no deletions. Sequences that mapped to multiple locationson the genome were ignored. All other mapped reads were counted assequence tags, and only loci to which 40 and 100 sequence tags weremapped were considered for further analysis i.e. only bases havingcoverage of 40 and 100 tags were considered.

For each base locus, the number of tags that mapped to each of the fourbases were counted. Loci having more than two possible bases wereeliminated, and only tags that mapped to monoallelic and biallelic lociwere used to predict the artificial fetal fraction. The total number oftags that mapped at each base locus represented the coverage (D) at thatlocus. In this simulated maternal sample, it is expected that thecontribution of the mother's major allele (B) would reflect the 90%portion of the tags, and the contribution of the son's minor allele (A)would reflect the 10% portion of the tags.

FIGS. 11 A and B show histograms of the number of variant baseobservations (Frequency) on chromosomes 1 and 7, respectively for minorallele percentages (A/D) for chromosomes 1 and 7. The percent minorallele is the percent of the total number of alleles at a given locus.For example, for a given locus at which there are 8 occurrences of minorallele A and 56 occurrences of major allele B, then the percent minorallele is 8%. The data show that the greatest number of occurrences(Frequency) for the minor allele are observed when the minor allele ispresent at 5%, which represents half of the fetal fraction. Accordingly,the data predicted that the sample contained a fetal fraction of 10%,which corresponds with that used to create the artificial maternalsample.

FIGS. 12A and B show the distribution of allelic frequency alongchromosomes 1 and 7, respectively. Both plots show the maximum number ofvariant alleles along the chromosomes occur at minor allele frequency of5% and major allele frequency of 95%. Some of the remaining data pointsrepresent biallelic loci present in the mother's genome, while othersrepresent noise of the sequencing methodology. The central portion ofeach plot where variant alleles are not represented coincides with thechromosome's centromeres, which are known to be repeat-rich regions ofchromosomes, to which tags map at more than one locus and are thereforeexcluded from the analysis. At other regions, for example regionsflanking the centromere and regions corresponding to telomeres, variantalleles are over-represented. Over-representation of these regions canbe attributed to the sequencing methodology whereby some regions aresequenced at greater levels than others.

Therefore, the present method can be used to predict fetal fraction. Themethod is particularly useful as it does not require identification oftargeted sequences e.g. SNPs, and any variation at any position of anychromosome can serve to predict the percent fetal fraction.

Other Embodiments

Although the above has generally described the present inventionaccording to specific processes and apparatus, the present invention hasa much broader range of applicability. In particular, the presentinvention has been described in terms of detecting the fraction of fetalDNA in a DNA sample taken from a pregnant individual, but is not solimited, as the concepts and methods presented herein may also beapplied in other contexts such as detecting the relative amounts of DNAtypes in a sample having DNA originating from two or more differentgenomes. Of course, those of ordinary skill in the art will recognizeother variations, modifications, and alternatives.

For example, although most of the examples and applications describedherein concern estimation of the fetal fraction of DNA in a DNA sampletaken from an individual carrying a fetus, the invention is not solimited. More generally, various embodiments provide for assessingrelative amounts of nucleic from two different genomes in a test samplethat contains a mixture of nucleic acids from the two different genomes,and which are known or are suspected to differ in the amount of one ormore sequence of interest. The mixture of nucleic acids is derived fromtwo or more types of cells.

Further, although most of the examples presented herein concern samplestaken from a pregnant human, the disclosure is not so limited. Forexample, the individual providing a sample to be tested can be anorganism comprising polynucleotide sequences, e.g., a plant, an insectsuch as a fly, or an animal. In some embodiments, the subject is amammal, e.g., a mouse, rat, dog, monkey or human. As indicted, thesubject can be a pregnant individual. The subject might be an individualwith a disease such as a cancer, or might be infected with a foreignbody such as a microorganism, e.g., a virus. The sample can comprise abodily fluid from the subject, e.g., blood, plasma, serum, sputum,saliva, urine, excrement, pus, lymph, mucous or the like. For example,the sample may be a maternal plasma sample containing a mixture ofmaternal and fetal cell free DNA. Generally, the disclosed methods mayinvolve sequencing DNA from a sample; mapping the sequence reads topolymorphisms; classifying the polymorphisms on the basis of zygosity;and estimating the fraction of DNA from a secondary source in thesample.

APPENDIX 1Allele Search Database Sequence Listing >rs560681.1|Chr.1|length =111|allele = A CACATGCACA GCCAGCAACC CTGTCAGCAG GAGTTCCCACCAGTTTCTTT CTGAGAACAT CTGTTCAGGT TTCTCTCCATCTCTATTTAC TCAGGTCACA GGACCTTGGG G >rs560681.2|Chr.1|length =111|allele = G CACATGCACA GCCAGCAACC CTGTCAGCAG GAGTTCCCACCAGTTTCTTT CTGAGAACAT CTGTTCAGGT TTCTCTCCATCTCTGTTTAC TCAGGTCACA GGACCTTGGG G >rs1109037.1|Chr.2|length =126|allele = A TGAGGAAGTG AGGCTCAGAG GGTAAGAAAC TTTGTCACAGAGCTGGTGGT GAGGGTGGAG ATTTTACACT CCCTGCCTCCCACACCAGTT TCTCCAGAGT GGAAAGACTT TCATCTCGCACTGGCA >rs1109037.2|Chr.2|length = 126|allele = GTGAGGAAGTG AGGCTCAGAG GGTAAGAAAC TTTGTCACAGAGCTGGTGGT GAGGGTGGAG ATTTTACACT CCCTGCCTCCCACACCAGTT TCTCCGGAGT GGAAAGACTT TCATCTCGCACTGGCA >rs9866013.1|Chr.3|length = 121|allele = CGTGCCTTCAG AACCTTTGAG ATCTGATTCT ATTTTTAAAGCTTCTTAGAA GAGAGATTGC AAAGTGGGTT GTTTCTCTAGCCAGACAGGG CAGGCAAATA GGGGTGGCTG GTGGGATGGGA >rs9866013.2|Chr.3|length =121|allele = T GTGCCTTCAG AACCTTTGAG ATCTGATTCT ATTTTTAAAGCTTCTTAGAA GAGAGATTGC AAAGTGGGTT GTTTCTCTAGCCAGACAGGG CAGGTAAATA GGGGTGGCTG GTGGGATGGGA >rs13182883.1|Chr.5|length =111|allele = A AGGTGTGTCT CTCTTTTGTG AGGGGAGGGG TCCCTTCTGGCCTAGTAGAG GGCCTGGCCT GCAGTGAGCA TTCAAATCCTCAAGGAACAG GGTGGGGAGG TGGGACAAAG G >rs13182883.2|Chr.5|length =111|allele = G AGGTGTGTCT CTCTTTTGTG AGGGGAGGGG TCCCTTCTGGCCTAGTAGAG GGCCTGGCCT GCAGTGAGCA TTCAAATCCTCGAGGAACAG GGTGGGGAGG TGGGACAAAG G >rs13218440.1|Chr.6|length =139|allele = A CCTCGCCTAC TGTGCTGTTT CTAACCATCA TGCTTTTCCCTGAATCTCTT GAGTCTTTTT CTGCTGTGGA CTGAAACTTGATCCTGAGAT TCACCTCTAG TCCCTCTGAG CAGCCTCCTGGAATACTCAG CTGGGATGG >rs13218440.2|Chr.6|length = 139|allele = GCCTCGCCTAC TGTGCTGTTT CTAACCATCA TGCTTTTCCCTGAATCTCTT GAGTCTTTTT CTGCTGTGGA CTGAAACTTGATCCTGAGAT TCACCTCTAG TCCCTCTGGG CAGCCTCCTGGAATACTCAG CTGGGATGG >rs4606077.1|Chr.8|length = 114|allele = CGCAACTCCCT CAACTCCAAG GCAGACACCA AAGCCCTCCCTGCCTGTGGC TTTGTAGTTC TAGTGTGGGA TCTGACTCCCCACAGCCCAC CCAAAGCCGG GGAACTCCTC ACTG >rs4606077.2|Chr.8|length =114|allele = T GCAACTCCCT CAACTCCAAG GCAGACACCA AAGCCCTCCCTGCCTGTGGC TTTGTAGTTC TAGTGTGGGA TCTGACTCCCCACAGCCTAC CCAAAGCCGG GGAACTCCTC ACTG >rs7041158.1|Chr.9|length =117|allele = C AATTGCAATG GTGAGAGGTT GATGGTAAAA TCAAACGGAACTTGTTATTT TGTCATTCTG ATGGACTGGA ACTGAGGATTTTCAATTTCC TCTCCAACCC AAGACACTTC TCACTGG >rs7041158.2|Chr.9|length =117|allele = T AATTGCAATG GTGAGAGGTT GATGGTAAAA TCAAACGGAACTTGTTATTT TGTCATTCTG ATGGACTGGA ACTGAGGATTTTCAATTTCC TTTCCAACCC AAGACACTTC TCACTGG >rs740598.1|Chr.10|length =114|allele = A GAAATGCCTT CTCAGGTAAT GGAAGGTTAT CCAAATATTTTTCGTAAGTA TTTCAAATAG CAATGGCTCG TCTATGGTTAGTCTCACAGC CACATTCTCA GAACTGCTCA AACC >rs740598.2|Chr.10|length =114|allele = G GAAATGCCTT CTCAGGTAAT GGAAGGTTAT CCAAATATTTTTCGTAAGTA TTTCAAATAG CAATGGCTCG TCTATGGTTAGTCTCGCAGC CACATTCTCA GAACTGCTCA AACC >rs10773760.1|Chr.12|length =128|allele = A ACCCAAAACA CTGGAGGGGC CTCTTCTCAT TTTCGGTAGACTGCAAGTGT TAGCCGTCGG GACCAGCTTC TGTCTGGAAGTTCGTCAAAT TGCAGTTAAGTCCAAGTATG CCACATAGCAGATAAGGG >rs10773760.2|Chr.12|length = 128|allele = GACCCAAAACA CTGGAGGGGC CTCTTCTCAT TTTCGGTAGACTGCAAGTGT TAGCCGTCGG GACCAGCTTC TGTCTGGAAGTTCGTCAAAT TGCAGTTAGG TCCAAGTATG CCACATAGCAGATAAGGG >rs4530059.1|Chr.14|length = 110|allele = AGCACCAGAAT TTAAACAACG CTGACAATAA ATATGCAGTCGATGATGACT TCCCAGAGCT CCAGAAGCAA CTCCAGCACACAGAGAGGCG CTGATGTGCC TGTCAGGTGC >rs4530059.2|Chr.14|length =110|allele = G GCACCAGAAT TTAAACAACG CTGACAATAA ATATGCAGTCGATGATGACT TCCCAGAGCT CCAGAAGCAA CTCCAGCACACGGAGAGGCG CTGATGTGCC TGTCAGGTGC >rs1821380.1|Chr.15|length =139|allele = C GCCCAGATTA GATGGAACCT TTTCCTCTTT TCCAGTGCAAGACAAGCGAT TGAAAGAAGT GGATGTGTTA TTGCGGGCACAATGGAGCCA CTGAACTGCA GTGCAAAAAT GCAGTAAGGCATACAGATAG AAGAAGGAG >rs1821380.2|Chr.15|length = 139|allele = GGCCCAGATTA GATGGAACCT TTTCCTCTTT TCCAGTGCAAGACAAGCGAT TGAAAGAAGT GGATGTGTTA TTGCGGGCACAATGGAGCCA CTGAACTGCA GTGCAAAAAT GCAGTAAGGGATACAGATAG AAGAAGGAG >rs7205345.1|Chr.16|length = 116|allele = CTGACTGTATA CCCCAGGTGC ACCCTTGGGT CATCTCTATCATAGAACTTA TCTCACAGAG TATAAGAGCT GATTTCTGTGTCTGCCTCTC ACACTAGACT TCCACATCCT TAGTGC >rs7205345.2|Chr.16|length =116|allele = G TGACTGTATA CCCCAGGTGC ACCCTTGGGT CATCTCTATCATAGAACTTA TCTCACAGAG TATAAGAGCT GATTTCTGTGTCTGCCTGTC ACACTAGACT TCCACATCCT TAGTGC >rs8078417.1|Chr.17|length =110|allele = C TGTACGTGGT CACCAGGGGA CGCCTGGCGC TGCGAGGGAGGCCCCGAGCC TCGTGCCCCC GTGAAGCTTC AGCTCCCCTCCCCGGCTGTC CTTGAGGCTC TTCTCACACT >rs8078417.2|Chr.17|length =110|allele = T TGTACGTGGT CACCAGGGGA CGCCTGGCGC TGCGAGGGAGGCCCCGAGCC TCGTGCCCCC GTGAAGCTTC AGCTCCCCTCCCTGGCTGTC CTTGAGGCTC TTCTCACACT >rs576261.1|Chr.19|length =114|allele = A CAGTGGACCC TGCTGCACCT TTCCTCCCCT CCCATCAACCTCTTTTGTGC CTCCCCCTCC GTGTACCACC TTCTCTGTCACCAACCCTGG CCTCACAACT CTCTCCTTTG CCAC >rs576261.2|Chr.19|length =114|allele = C CAGTGGACCC TGCTGCACCT TTCCTCCCCT CCCATCAACCTCTTTTGTGC CTCCCCCTCC GTGTACCACC TTCTCTGTCACCACCCCTGG CCTCACAACT CTCTCCTTTG CCAC >rs2567608.1|Chr.20|length =110|allele = A CAGTGGCATA GTAGTCCAGG GGCTCCTCCT CAGCACCTCCAGCACCTTCC AGGAGGCAGC AGCGCAGGCA GAGAACCCGCTGGAAGAATC GGCGGAAGTT GTCGGAGAGG >rs2567608.2|Chr.20|length =110|allele = A CAGTGGCATA GTAGTCCAGG GGCTCCTCCT CAGCACCTCCAGCACCTTCC AGGAGGCAGC AGCGCAGGCA GAGAACCCGCTGGAAGGATC GGCGGAAGTT GTCGGAGAGG >rs2073383.1|Chr.22|length =140|allele = C GCTGCAGAAT CCACAGAGCC AGACGCCCCC TGGGCCCCCAGCGCCCCCCT GCACAAGTGG GGAAACTAGG TCATGGGGCCCAGGCAGTGT GGAAGGCGTT GCAGGAGTTG CCCAGGGCGTGGGGTCCTCC AGCCTCAGTG >rs2073383.2|Chr.22|length = 140|allele = TGCTGCAGAAT CCACAGAGCC AGACGCCCCC TGGGCCCCCAGCGCCCCCCT GCACAAGTGG GGAAACTAGG TCATGGGGCCCAGGCAGTGT GGAAGGCGTT GCAGGAGTTG CCCAGGGTGT GGGGTCCTCC AGCCTCAGTG

1. A method of estimating the fraction of fetal DNA in DNA obtained froma bodily fluid of a pregnant individual, the method comprising: (a)receiving a sample of the bodily fluid; (b) extracting DNA from thesample under conditions that extract DNA of both a maternal genome and afetal genome present in the bodily fluid; (c) sequencing the extractedDNA with a nucleic acid sequencer under conditions that produce DNAsegment sequences containing one or more polymorphisms; (d) mapping theDNA segment sequences derived from sequencing the DNA in the bodilyfluid to one or more designated polymorphisms on a reference sequence,wherein the mapping is performed using a computational apparatusprogrammed to map nucleic acid sequences to the one or more designatedpolymorphisms; (e) determining allele frequencies of the mapped DNAsegment sequences for at least one of the designated polymorphisms; (f)classifying the at least one designated polymorphism based on acombination of the zygosity of the pregnant individual and the zygosityof the fetus; and (g) estimating the fraction of fetal DNA in the DNAobtained from the pregnant individual using the allele frequenciesdetermined in (e) and the combination of zygosities from (f), wherein(e)-(g) are performed on one or more processors running under programinstructions for the determining, classifying and estimating.
 2. Themethod of claim 1, wherein the classifying in (f) classifies the atleast one designated polymorphism into one of the followingcombinations: (i) the pregnant individual is homozygous and the fetus ishomozygous, (ii) the pregnant is individual homozygous and the fetus isheterozygous, (iii) the pregnant individual is heterozygous and thefetus is homozygous, and (iv) the pregnant individual is heterozygousand the fetus is heterozygous.
 3. The method of claim 2, furthercomprising removing from consideration any polymorphism classified incombination (i) or combination (iv).
 4. The method of claim 1, furthercomprising filtering the at least one designated polymorphisms to removefrom consideration any polymorphism having a minor allele frequency ofgreater than a defined threshold.
 5. The method of claim 1, furthercomprising filtering the at least one designated polymorphisms to removefrom consideration any polymorphism having a minor allele frequency ofless than a defined threshold.
 6. The method of claim 1, whereinclassifying the at least one designated polymorphism comprises applyinga threshold to the allele frequency determined in (e).
 7. The method ofclaim 1, wherein classifying the at least one designated polymorphismcomprises applying the allele frequency data from (e), obtained for aplurality of polymorphisms, to a mixture model.
 8. The method of claim7, wherein the mixture model employs factorial moments.
 9. The method ofclaim 1, wherein the DNA obtained from a bodily fluid of a pregnantindividual is cell-free DNA obtained from the plasma of the pregnantindividual.
 10. The method of claim 1, wherein mapping the DNA segmentsobtained from the blood of the individual carrying the fetus comprisescomputationally mapping said segments to a database of polymorphisms.11. The method of claim 1, wherein the sequencing is conducted withoutselectively amplifying any of the one or more designated polymorphisms.12. The method of claim 1, further comprising executing programinstructions on the one or more processors to automatically record thefraction of fetal of DNA as estimated in (g) in a patient medicalrecord, stored on a computer readable medium, for the pregnantindividual.
 13. The method of claim 12, wherein said patient medicalrecord is maintained by a laboratory, physician's office, a hospital, ahealth maintenance organization, an insurance company, or a personalmedical record website.
 14. The method of claim 1, further comprising,based on the estimate of the fraction of fetal DNA, prescribing,initiating, and/or altering treatment of a human subject from whom thematernal test sample was taken.
 15. The method of claim 1, furthercomprising, based on the estimate of the fraction of fetal DNA, orderingand/or performing one or more additional tests.
 16. An apparatus forestimating the fraction of fetal DNA in DNA obtained from a bodily fluidof a pregnant individual, the apparatus comprising: (a) a sequencerconfigured to (i) receive DNA extracted from a sample of the bodilyfluid comprising DNA of both a maternal genome and a fetal genome, and(ii) sequence the extracted DNA under conditions that produce DNAsegment sequences containing one or more designated polymorphisms; and(b) a computational apparatus configured to instruct one or moreprocessors to map nucleic acid sequences to the one or more designatedpolymorphisms on a reference sequence, determine allele frequencies ofthe mapped DNA segment sequences for at least one of the designatedpolymorphisms, classify the at least one designated polymorphism basedon a combination of the zygosity of the pregnant individual and thezygosity of the fetus, and estimate the fraction of fetal DNA in the DNAobtained from the pregnant individual using the allele frequencies andthe combination of zygosities.
 17. The apparatus of claim 16, furthercomprising a tool for extracting DNA from the sample under conditionsthat extract DNA of both the maternal genome and the fetal genome. 18.The apparatus of claim 16, wherein the computational apparatus isfurther configured to instruct the one or more processors to classifythe at least one designated polymorphism into one of the followingcombinations: (i) the pregnant individual is homozygous and the fetus ishomozygous, (ii) the pregnant is individual homozygous and the fetus isheterozygous, (iii) the pregnant individual is heterozygous and thefetus is homozygous, and (iv) the pregnant individual is heterozygousand the fetus is heterozygous.
 19. The apparatus of claim 18, whereinthe computational apparatus is further configured to instruct the one ormore processors to remove from consideration any polymorphism classifiedin combination (i) or combination (iv).
 20. The apparatus of claim 16,wherein the computational apparatus is further configured to instructthe one or more processors to remove from consideration any polymorphismhaving a minor allele frequency of greater than a defined threshold. 21.The apparatus of claim 16, wherein the computational apparatus isfurther configured to instruct the one or more processors to filter theone or more designated polymorphisms to remove from consideration anypolymorphism having a minor allele frequency of less than a definedthreshold.
 22. The apparatus of claim 16, wherein the computationalapparatus is further configured to instruct the one or more processorsto classify the at least one designated polymorphism by applying athreshold to the allele frequency.
 23. The apparatus of claim 16,wherein the computational apparatus is further configured to instructthe one or more processors to classify the at least one designatedpolymorphism by applying the allele frequency data, obtained for aplurality of polymorphisms, to a mixture model.
 24. The apparatus ofclaim 23, wherein the mixture model employs factorial moments.
 25. Theapparatus of claim 16, further comprising apparatus for extractingcell-free DNA obtained from the plasma of the pregnant individual forsequencing in the sequencer.
 26. The apparatus of claim 16, furthercomprising a database of polymorphisms, wherein the computationalapparatus is further configured to instruct the one or more processorsto map the DNA segments obtained from the blood of the individualcarrying the fetus by computationally mapping said segments to thedatabase of polymorphisms.
 27. The apparatus of claim 16, wherein thecomputational apparatus is further configured to instruct the one ormore processors to automatically record the fraction of fetal of DNA ina patient medical record, stored on a computer readable medium, for thepregnant individual.
 28. The apparatus of claim 27, wherein said patientmedical record is maintained by a laboratory, physician's office, ahospital, a health maintenance organization, an insurance company, or apersonal medical record website.
 29. A method of estimating a fractionof fetal DNA in DNA obtained from a bodily fluid of a pregnantindividual, the method comprising: (a) mapping segments of DNA obtainedfrom the bodily fluid of the pregnant individual to a plurality ofpolymorphism sequences, wherein the DNA was sequenced under conditionsthat identify the plurality of polymorphism sequences; (b) determiningan allele frequency of the mapped nucleic acids for each of theplurality polymorphism sequences; and (c) applying the allelefrequencies to a mixture model to obtain an estimate of the fraction offetal DNA in the DNA obtained from the blood of the individual carryingthe fetus, wherein (b)-(c) are performed on one or more processorsrunning under program instructions for the determining and applying. 30.The method of claim 29, wherein (c) comprises executing instructions onthe one or more processors for solving a series of equations forfactorial moments of allele frequency data for each of the plurality ofpolymorphism sequences.
 31. The method of claim 29, further comprising,prior to (c), computationally removing allele frequencies forpolymorphisms identified has being heterozygous in both the fetus andthe pregnant individual.
 32. The method of claim 29, further comprising,prior to (c), computationally removing allele frequencies forpolymorphisms identified has being homozygous in both the fetus and thepregnant individual.
 33. The method of claims 29, further comprising,prior to (c), computationally removing allele frequencies forpolymorphisms identified has being heterozygous in the pregnantindividual.
 34. The method of claim 29, wherein the mixture modelaccounts for sequencing error.
 35. The method of claim 29, wherein theDNA obtained from a bodily fluid of a pregnant individual is cell-freeDNA obtained from the plasma of the pregnant individual.
 36. The methodof claim 29, wherein mapping the nucleic acids obtained from bodilyfluid of a pregnant individual comprises mapping said segments to adatabase of polymorphisms.
 37. The method of claim 29, furthercomprising obtaining a sample of the bodily fluid of the pregnantindividual.
 38. The method of claim 29, further comprising sequencingthe DNA from the bodily fluid of pregnant individual with a nucleic acidsequencer under conditions that produce DNA segment sequences containingthe polymorphism sequences.
 39. The method of claim 29, wherein themapping in (a) comprises identifying a plurality of biallelicpolymorphism sequences.
 40. The method of claim 29, wherein the mappingin (a) comprises mapping the segments of DNA to a plurality ofpre-defined polymorphism sequences.
 41. The method of claim 29, furthercomprising executing program instructions on the one or more processorsto automatically record the fraction of fetal of DNA as estimated in (c)in a patient medical record, stored on a computer readable medium, forthe pregnant individual.
 42. The method of claim 41, wherein saidpatient medical record is maintained by a laboratory, physician'soffice, a hospital, a health maintenance organization, an insurancecompany, or a personal medical record website.
 43. The method of claim29, further comprising, based on the estimate of the fraction of fetalDNA, prescribing, initiating, and/or altering treatment of a humansubject from whom the maternal test sample was taken.
 44. The method ofclaim 29, further comprising, based on the estimate of the fraction offetal DNA, ordering and/or performing one or more additional tests. 45.A method of estimating the fraction of fetal DNA in DNA obtained from abodily fluid of a pregnant individual, the method comprising: (a)receiving a sample of the bodily fluid; (b) extracting DNA from thesample under conditions that extract DNA of both a maternal genome and afetal genome present in the bodily fluid; (c) sequencing the extractedDNA with a nucleic acid sequencer under conditions that produce DNAsegment sequences; (d) comparing the DNA segment sequences derived fromthe bodily fluid and from the comparison identifying one or morebiallelic polymorphisms, wherein the comparing is performed using acomputational apparatus programmed to compare nucleic acid sequences andidentify polymorphisms; (e) determining allele frequencies of the DNAsegment sequences for at least one of the identified polymorphisms; (f)classifying the at least one identified polymorphism based on acombination of the zygosity of the pregnant individual and the zygosityof the fetus; and (g) estimating the fraction of fetal DNA in the DNAobtained from the pregnant individual using the allele frequenciesdetermined in (e) and the combination of zygosities from (f), wherein(e)-(g) are performed on one or more processors running under programinstructions for the determining, classifying and estimating.
 46. Themethod of claim 45, wherein the classifying in (f) classifies the atleast one identified polymorphism into one of the followingcombinations: (i) the pregnant individual is homozygous and the fetus ishomozygous, (ii) the pregnant is individual homozygous and the fetus isheterozygous, (iii) the pregnant individual is heterozygous and thefetus is homozygous, and (iv) the pregnant individual is heterozygousand the fetus is heterozygous.
 47. The method of claim 46, furthercomprising removing from consideration any polymorphism classified incombination (i) or combination (iv).
 48. The method of claim 45, furthercomprising filtering the one or more identified polymorphisms to removefrom consideration any polymorphism having a minor allele frequency ofgreater than a defined threshold.
 49. The method of claim 45, whereinthe DNA segment sequences have a length of between about 20 base pairsand about 300 base pairs.
 50. The method of claim 45, whereinclassifying the at least one identified polymorphism comprises applyinga threshold to the allele frequency determined in (e).
 51. The method ofclaim 45, wherein classifying the at least one identified polymorphismcomprises applying the allele frequency data from (e), obtained for aplurality of polymorphisms, to a mixture model.
 52. The method of claim51, wherein the mixture model employs factorial moments.
 53. The methodof claim 45, wherein the DNA obtained from a bodily fluid of a pregnantindividual is cell-free DNA obtained from the plasma of the pregnantindividual.
 54. An apparatus for estimating a fraction of fetal DNA inDNA obtained from a bodily fluid of a pregnant individual, the apparatuscomprising: (a) a sequencer configured to (i) receive DNA extracted froma sample of the bodily fluid comprising DNA of both a maternal genomeand a fetal genome, and (ii) sequence the extracted DNA to producesequence segments of DNA; and (b) a computational apparatus configuredto instruct one or more processors to map the sequence segments of DNAobtained from the bodily fluid of the pregnant individual to a pluralityof polymorphism sequences, determine an allele frequency for each of theplurality of polymorphism sequences from the mapped sequence segments ofDNA, and apply the allele frequencies to a mixture model to obtain anestimate of the fraction of fetal DNA in the DNA obtained from the bloodof the individual carrying the fetus.
 55. An apparatus for estimatingthe fraction of fetal DNA in DNA obtained from a bodily fluid of apregnant individual, the apparatus comprising: (a) a sequencerconfigured to (i) receive DNA extracted from a sample of the bodilyfluid comprising DNA of both a maternal genome and a fetal genome, and(ii) sequence the extracted DNA under conditions that produce DNAsegment sequences; and (b) a computational apparatus configured toinstruct one or more processors to compare the DNA segment sequencesderived from the bodily fluid and from the comparison identifying one ormore biallelic polymorphisms, determine allele frequencies of the DNAsegment sequences for at least one of the identified polymorphisms,classify the at least one identified polymorphism based on a combinationof the zygosity of the pregnant individual and the zygosity of thefetus, and estimate the fraction of fetal DNA in the DNA obtained fromthe pregnant individual using the allele frequencies and the combinationof zygosities.